Journal articles: 'MEL cells; Epigenetic marks'

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Black, Kathryn, Elena Sotillo, Nicole Martinez, Matthew Gazzara, Alejandro Barrera, Yoseph Barash, Kristen Lynch, and Andrei Thomas-Tikhonenko. "Regulation of CD19 Exon 2 Inclusion in B-Lymphoid Cells By Splicing Factors and Epigenetic Marks." Blood 126, no. 23 (December 3, 2015): 2425. http://dx.doi.org/10.1182/blood.v126.23.2425.2425.

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Abstract CD19 is expressed broadly on the surface of B-cells during normal development and malignant growth, making it a good target for immunotherapy. While immunotherapies targeting CD19 have had great success against pediatric B-cell acute lymphoblastic leukemia (B-ALL), relapses lacking the CD19 epitope still occur (Maude et al., 2014). We have discovered that alternative splicing of CD19, in particular the skipping of exon 2, is responsible for the loss of CD19 extracellular domains, causing resistance to therapy (Sotillo et al., 2015). Here we investigate the molecular mechanism of CD19 exon 2 skipping. The sequence-based algorithm AVISPA (Barash et al., 2013) predicts several splicing factors (SF) to bind near exon 2. We used RNA crosslink immunoprecipitation (CLIP) in nuclear lysates from Nalm-6 B-ALL cells to test the direct binding to exon 2 of 9 AVISPA-predicted SFs and 6 SFs commonly involved in exon skipping. This allowed us to identify SRSF3, hnRNP-A, and hnRNP-C as CD19 exon 2-bound proteins. Subsequent siRNA knockdown experiments reveled that downregulation of SRSF3, but not hnRNP-C, increases the frequency of exon 2 skipping in a dose dependent manner, suggesting that SRSF3 promotes the inclusion of exon 2. To further validate the role of SRSF3 in CD19 splicing we mined the publicly available GSE52834 dataset where 22 RNA binding proteins were knocked down in the GM19238 lymphoblastoid cell line. Of all siRNAs tested, only the anti-SRSF3 siRNA caused an increase in exon 2 skipping, suggesting that SRSF3 is indeed the key regulator of CD19 splicing. Interestingly, SRSF3 has been shown to interact with PSIP1, a cofactor known to "read" modified histone H3K36me3 (Pradeepa et al., 2012), suggesting a convergence of splicing-based and epigenetics mechanisms. Indeed, exonic regions in genomic DNA are enriched for H3K36me3, and knockdown of Setd2, the H3K36 methyltransferase, results in changes in exon inclusion (Luco et al., 2010; Brown et al., 2012; Hnilicova and Stanek, 2011). Thus, we are currently investigating the connection between the H3K36me3 marks in the CD19 locus and alternative splicing of CD19. Our data could suggest a method of restoring full-length CD19 expression in immunotherapy-resistant cancers using epigenetic drugs. Maude, S L, Noelle, F, Shaw, PA, Aplenc, R, Barrett, DM, Bunin, NJ, Chew, A, Gonzalez, VE, Zheng, Z, Lacey, SF, Mahnke, YD, Melenhorst, JJ, Rheingold, SR, Shen, A, Teachey, DT, Levine, BL, June CH, Porter, DL, and Grupp, SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507-1517. Sotillo, E, Barrett, D, Bagashev, A, Black, K, Lanauze, C, Oldridge, D, Sussman, R, Harrington, C, Chung, EY, Hofmann, TJ, Maude, SL, Martinez, NM, Raman, P, Ruella, M, Allman, D, Jacoby, E, Fry, T, Barash, Y, Lynch, KW, Mackall, C, Maris, J, Grupp, SA, and Thomas-Tikhonenko, A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015AACR Annual Meeting, Philadelphia. Barash Y, Vaquero-Garcia J, González-Vallinas J, Xiong HY, Gao W, Lee LJ, and Frey BJ. AVISPA: a web tool for the prediction and analysis of alternative splicing. Genome Biol 2013; 14(10):R114. Pradeepa, MM, Sutherland, HG, Ule, J, Grimes, GR, and Bickmore, WA. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLOS Genets 2012; 8:e1002717. Luco, RF, Pan, Q, Tominaga, K, Blencowe, BJ, Pereira-Smith, OM, Misteli, T. Regulation of alternative splicing by histone modifications. Science 2010; 327: 996-1000. Brown, SJ, Stoilov, P, and Xing, Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human Mol Genets 2012; 21:R90-R96. Hnilicova, J, and Stanek, D. Where splicing joins chromatin. Nucleus 2011; 2:182-188. Disclosures No relevant conflicts of interest to declare.

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Karkhanis, Vrajesh, Lapo Alinari, Bethany Mundy, Michael Caliguri, Selina Chen-Kiang, Olivier Elemento, Sif Said, and Robert A. Baiocchi. "PRMT5 Targets Tumor Suppressor Micro RNAs to Regulate Cyclin D1 and c-MYC in Mantle Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 2937. http://dx.doi.org/10.1182/blood.v128.22.2937.2937.

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Abstract Protein arginine methyltransferase-5 (PRMT5), a major type II arginine methyltransferase, is an important epigenetic modifier with oncogene-like properties due to its transcriptional repressive activity. When over-expressed, PRMT5 has been shown to target and silence the expression of multiple regulatory and tumor suppressor genes. Global symmetric dimethylation of arginine residues within the N-terminal of histones (H2A(Me2)R3, H3(Me2)R8, H4(Me2)R3) plays a critical role in B cell transformation, correlates with increased tumor cell proliferation and survival. PRMT5 expression is enhanced in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma (MCL) and supports constitutive CYCLIN D1/CDK4/6 activity leading to inactivation of the RBL2/E2F tumor suppressor pathway. Other work has identified PRMT5 as a vital contributor to MYC-driven oncogenesis. While PRMT5 has been characterized as a transcriptional repressor, few micro-RNAs (miRs) have been identified as direct targets. Here we utilize next generation sequencing and whole genome mapping to identify miRs targeted by PRMT5 in mantle cell lymphomas. ChIP-seq analysis revealed genome-wide recruitment of the PRMT5-specific epigenetic mark H3(Me2)R8 in B-cell lymphoma cell lines (Jeko, Pfeiffer, SUDHL2) with minimal enrichment on chromatin from normal B cells. PRMT5 was found to target as many as 8593 genes in each lymphoma cell line examined with 4662 genes that are common targets between three different lymphoma cell lines. Comparing Chip-Seq to RNA-Seq data of each lymphoma cell line treated with PRMT5 shRNA identified miR-33b, miR-96 and miR-503 as direct targets. Predicted 3' untranslated region (UTR) targets of these miRs included CYCLIN D1 (miR-33b, miR-96, miR-503), MYC (miR-33b) and PRMT5 (miR-96), three gene products that have been shown to be highly relevant to the malignant phenotype of aggressive MCL. Validation studies with ChIP-real-time polymerase chain reaction (RT-PCR) and quantitative-RT-PCR demonstrated that CRISPR-CAS9-mediated PRMT5 deletion led to transcriptional derepression of miR-33b, miR-96 and miR-503 in MCL lines (CC-MCL, Jeko, and SP53) and in primary MCL patient samples. Inhibition of PRMT5 led to loss of recruitment of an epigenetic repressor complex at the miR-96 promoter containing PRMT5 and HDAC3, gain of p65 and enrichment of hyperacetylated lysine epigenetic marks H4K8, H3K14 and H2BK12, changes consistent with restored transcriptional activity of miR96. Promoter regions of miR33b and miR503 showed loss of recruitment of a repressive complex consisting of SP1, HDAC2 and gain of p300, CBP and hyperacetylated lysine epigenetic marks H3K9 and H3K14 following PRMT5 inhibition. Restored expression of miR-33b led to simultaneous down-modulation of CYCLIN D1 and c-MYC, whereas miR-96 re-expression led to loss of CYCLIN D1 and PRMT5. Re-expression of miR-503 on the contrary only impacted CYCLIN D1 expression. Furthermore, luciferase reporter assays with wild-type and mutant 3' UTRs showed that binding sites of miR-33b, miR-96 and miR-503 were critical for translational regulation of CYCLIN D1 and C-MYC. Inducible re-expression of miR-33b, miR-96 and miR-503 inhibited proliferation of MCL cells as determined by MTS assay and promoted cellular apoptosis as measured by staining with Annexin V/PI staining and flow cytometry. In vivo studies with the MCL cell line CC-MCL1 designed to conditionally express miR-96 and or miR-33b are currently underway. These results link dysregulated PRMT5 expression to transcriptional silencing of tumor suppressor miRs capable of regulating critical drivers of aggressive histologic subtypes of MCL and indicate that multiple mechanisms are involved in the regulation of CYCLIN D1 expression in MCL. Furthermore, this data supports PRMT5 overexpression as a critical factor involved with maintenance of the malignant phenotype of MCL, and supports strategies to selectively inhibit this promising therapeutic target in this disease. Disclosures Baiocchi: Essanex: Research Funding.

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Arumugam, Paritha, Fabrizia Urbinati, Chinavenmeni S. Velu, H. Leighton Grimes, and Punam Malik. "The 3′ End of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to the 5′ Insulator Core and Is Necessary in Conjunction with the Core for Full Insulator Activity." Blood 112, no. 11 (November 16, 2008): 817. http://dx.doi.org/10.1182/blood.v112.11.817.817.

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Abstract Genetic correction of hematologic defects is currently impeded by inefficient vector technology. We find that vectors that insulate the correcting transgene from position effects and genotoxicity compromise viral titers. Here we present an improved vector system which utilizes a modified insulator element, without sacrificing viral titers. Specifically, our genetic and epigenetic analysis of the 1.2kb chicken β-globin hypersensitive site-4 (cHS4) insulator reveal heretofore unknown activities in regions of the chicken β-globin insulator element outside the canonical and well studied 250bp 5′ “core” element. Previously, the core insulator activity was mapped to CTCF and USF-1/2 binding sites, located only in the 5′ 250bp core. However, we find that the 5′ 250bp core alone is ineffective at shielding from position effects when it flanks transgenes, and gammaretrovirus/lentivirus vectors. In contrast, the entire 1.2kb cHS4 efficiently insulates, but significantly lowers titers of lentiviral vectors. To identify insulating activities which might be appended to the 5′ 250bp core to properly insulate transgene expression cassettes without sacrificing viral titers, we performed a structure-function analysis of the cHS4 insulator placed within the 3′LTR of a lentivirus containing the regulatory and coding sequences of human b-globin (Table 1). We compared single-copy clonal progeny of mouse erythroleukemia cells (MEL) and primary transduced and transplanted hematopoietic stem cells for position effects. Additionally, we studied repressive and activating histone marks over the transgene promoter and cHS4 in the different proviruses. Our data indicate that while all vectors containing the core reduced the coefficient of variation (CV) of human b-globin (HbA) expression, several constructs suggested that cHS4 sequences in the most 3′ 400bp (furthest from the core) may be critical to full length insulator activity. We next analyzed HbA expression in vector-corrected thalassemia mice, and generated single copy secondary CFU-S, the gold standard for studying chromatin position effects (Table 2). While all vectors containing the cHS4 core provided some ‘insulator’ activity when compared to the uninsulated vector control (conceivably by reducing the CV/clonal variegation) the full length 1.2kb insulator vector provided maximum shielding from position effects, with nearly 2.5-fold higher HbA expression compared to the uninsulated vector. These data were confirmed in secondary CFU-S. Epigenetic analyses of the vector b-globin promoter revealed that transcriptionally repressive histone modifications were decreased, and activating histone modifications increased when the last 400bp sequences of cHS4 were present. Notably, vectors carrying only the 3′ 400bp sequences of cHS4 reduced clonal variegation in MEL cells and secondary CFU-S, but did not increase HbA expressing cells both in vitro and in vivo (Table 2). However, full insulator activity was restored in MEL clones when both the 5′ 250bp core was combined with the 3′ 400bp element. The addition of the 3′ 400bp element to the core was accompanied with a significant enrichment of active histone marks and minimal repressive histone marks in the provirus as seen with the 1.2Kb insulator. These data consolidate the known insulating activity of the 5′ 250bp core element with a novel 3′ 400bp element which (together) constitutes a new insulated vector system with excellent insulating properties and viral titers. Our data have important implications in the design of gene therapy vectors, where optimal insulator activity can be achieved with a minimal reduction in viral titers. Table 1. Single copy MEL clones showing effect of insulator sequences on position effects in β-globin carrying lentiviruses MEL Clones Uninsulated 5′ 250bp Core 5′ 400bp 2 Cores 5′800bp 1.2Kb 3′ 400bp HbA+ cells (%) 59±5 49±6 51±3 52±3 49±6 84±3** 59±3 CV of HbA expression 53±4 37±3* 40±2* 42±1* 42±3 37±1* 38±1* Table 2. Effect of insulator sequences on the differentiated progeny of transduced and transplanted thalassemia hematopoietic stem cells Primary transplants (24 wks) Mock Uninsulated 5′250 bp 5′400bp 2 cores 1.2Kb 3′ 400bp *P<0.05; **P<0.01; ***P<0.001; HbA = human β and mouse α globin tetramers Statistical analysis was performed by ANOVA (Dunnell’s multiple comparison test to the control vector sBG) *Data has not been nomialized far vector copies; CFU-S represent those screened for carrying single integrants by qPCR Note: Copy number of vector with 1.2kb cHS4 insert is significantly lower compared to uninsulated vector ^RBC (M/μl) 6.6±0.4 8.6±0.5 8.5±0.3 7.9±0.2 7.5±0.2 8.9±0.2 7.9±0.2 ^Hematecrit (%) 24±2 32±3 36±1 36±1 35±2 38±1 34±1 ^MCHC (g/dL) 24±1 20±1 29±2 29±2 27±2 33±1* 28±1 ^Reticulocyte (%) 29±2 11±4 10±2 12±7 11±13 7±1 12±2 Vector Copy (VC)/Cell 0 1.2±0.18 0.90±0.12 0.97±0.13 1.2±0.26 0.63±0.10 0.76±0.16 HbA (%)/VC (HPLC) 19±6 26±4 30±3 22±3 43±3** 18±3 HbA+ RBC/VC (FACS) 40±8 56±5 62±7 51±9 100±5** 32±7 ^CV 618±171 375±36* 384±29* 294±29** 281±16** 270±44** Secondary CFU-S (single copy) ^HbA+cells/CFUS (%) 37±4 53±4 53±5 42±4 85±2** 8±1*** ^CV 98±15 70±5* 75±3* 91±5 57±2** 59±2**

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Watanabe, Toshiki. "Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1–infected T cells." Blood 129, no. 9 (March 2, 2017): 1071–81. http://dx.doi.org/10.1182/blood-2016-09-692574.

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Abstract Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) that develops through a multistep carcinogenesis process involving 5 or more genetic events. We provide a comprehensive overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of genetic abnormalities in ATL that include alterations highly enriched in genes for T-cell receptor–NF-κB signaling such as PLCG1, PRKCB, and CARD11 and gain-of function mutations in CCR4 and CCR7. Conversely, the epigenetic landscape of ATL can be summarized as polycomb repressive complex 2 hyperactivation with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27 methyltransferase enhancer of zeste 2 was shown to be induced by HTLV-1 Tax and NF-κB. Furthermore, provirus integration site analysis with high-throughput sequencing enabled the analysis of clonal composition and cell number of each clone in vivo, whereas multicolor flow cytometric analysis with CD7 and cell adhesion molecule 1 enabled the identification of HTLV-1–infected CD4+ T cells in vivo. Sorted immortalized but untransformed cells displayed epigenetic changes closely overlapping those observed in terminally transformed ATL cells, suggesting that epigenetic abnormalities are likely earlier events in leukemogenesis. These new findings broaden the scope of conceptualization of the molecular mechanisms of leukemogenesis, dissecting them into immortalization and clonal progression. These recent findings also open a new direction of drug development for ATL prevention and treatment because epigenetic marks can be reprogrammed. Mechanisms underlying initial immortalization and progressive accumulation of these abnormalities remain to be elucidated.

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Chung, Jihyun, Vrajesh Karkhanis, Said Sif, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Supports MYC, Survivin and Cyclin D1 Activity in Aggressive Lymphomas By Regulating the WNT/β-Catenin Pathway." Blood 124, no. 21 (December 6, 2014): 58. http://dx.doi.org/10.1182/blood.v124.21.58.58.

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Abstract Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.

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Meng, Fanli, Kathrin Stamms, Romina Bennewitz, Andria Green, Fleur Oback, Pavla Turner, Jingwei Wei, and Björn Oback. "Targeted histone demethylation improves somatic cell reprogramming into cloned blastocysts but not postimplantation bovine concepti†." Biology of Reproduction 103, no. 1 (April 21, 2020): 114–25. http://dx.doi.org/10.1093/biolre/ioaa053.

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Abstract Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

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Chung, JI Hyun, Shelby Sloan, Peggy Scherle, Kris Vaddi, Said Sif, Rosa Lapalombella, and Robert A. Baiocchi. "PRMT5 Is a Key Epigenetic Regulator That Promotes Transcriptional Activation in Mantle Cell Lymphoma By Regulating the Lysine Methyltransferase SETD7 and MLL1 Activity." Blood 134, Supplement_1 (November 13, 2019): 2777. http://dx.doi.org/10.1182/blood-2019-131020.

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Introduction: Post-translational histone modifications directly modify chromatin structure to influence a wide variety of cellular events including gene expression, DNA replication and repair, and cell cycle control. While histone lysine methylation can confer either transcriptionally active or repressive states, the symmetric dimethylation of arginine residues on histone tails is generally associated with transcriptional repression. Overexpression and dysregulation of PRMT5, the major type II protein arginine methyltransferase, has been shown to drive cellular proliferation and survival of multiple cancer types including mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma associated with poor prognosis. Our work has shown that PRMT5 drives the symmetric dimethylation of arginine (histones H3R8 and H4R3) and attenuates RB/E2F regulatory pathways leading to the expression and activation of the PRC2 lysine methylation programs. We have previously shown that PRMT5 promotes constitutive expression of the WNT/β-CATENIN target genes (MYC, CCND1, and SURVIVIN) via the epigenetic repression of AXIN2 and WIF1 regulatory genes. This PRMT5 mediated epigenetic program allows for constitutive activation of WNT and PI3/AKT downstream pathways relevant to MCL growth and survival. The inhibition of PRMT5 triggers histone deacetylation and H3K4me3 demethylation on promoters of β-CATENIN target genes. H3K4 is modified by several enzymes containing SET domains including the SETD7 and MLL1 proteins. We hypothesized that PRMT5 inhibition, through its ability to repress AKT phosphorylation, would regulate SETD7 and MLL1 activity and regulate downstream pathways relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived MCL cell lines and primary lymphoma tumor cells was achieved with sh-PRMT5 lentivirus (or sh-GFP control) and utilization of a selective small molecule, SAM-competitive PRMT5 inhibitor (PRT382). Gene and protein expression was monitored by reverse transcription (RT) real time PCR and western immunoblotting, respectively. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. Interactions within the transcriptional complex were examined by co-immunoprecipitation. Cellular growth and apoptosis was assessed by proliferation assays and FACS analysis. Results: Inhibition of PRMT5 by shRNA-mediated knock down or treatment with PRT382 (Prelude Therapeutics) reduced H3K4me2 in MCL cells via indirect epigenetic repression of SETD7. ChIP-PCR studies showed PRMT5 to be recruited to the SETD7 promoter, suggesting that the activity of PRMT5 promoted the transcription of this lysine methyltransferase. Our findings show that reduced phosphor-AKT by PRMT5 inhibition inhibits dimerization of the MLL1 complex leading to dissociation of MLL1 from the transcriptional activation complex and decreased H3K4me1 and H3K4me3. PRMT5 inhibition led to dissociation of the BCL9- Pygopus-MLL1 transcriptional activating complex and to assembly of repressive LSD1 (the histone demethylase affecting H3K4me3)-HDAC2 (the histone lysine deacetylase) containing complexes. Furthermore, PRMT5 inhibition led to differentially enhanced recruitment of this repressive LSD1/HDAC2 complex and decreased H3K9Ac and H3K14Ac epigenetic marks on promoters of β-CATENIN target genes. PRMT5 inhibition regulates lysine methylation at H3K4 through epigenetic silencing of SETD7 and MLL1 activity, as well as histone H3 acetylation driven by histone acetyltransferase KAT2A. ChIP-Seq studies examining SETD7 and PRC2 recruitment in context of PRMT5 inhibition are currently underway. Conclusions: Our observations show that dysregulated PRMT5 activity acts as a master epigenetic regulator affecting arginine and lysine histone marks. PRMT5 can act directly to repress target tumor suppressor genes while simultaneously indirectly activating SETD7 and MLL1 to drive transcriptional activation of key oncogenes that promote the proliferation and survival of MCL. These results support the notion that inhibiting PRMT5 leads to erasure of repressive histone arginine and lysine marks and promote the restoration of normal growth and survival checkpoints in this disease. Disclosures Scherle: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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Lichtenberg, Jens, Elisabeth F. Heuston, Cheryl A. Keller, Ross C. Hardison, and David M. Bodine. "Comparison of Expression and Epigenetic Profiles in Human and Mouse Erythropoiesis and Megakaryopoiesis Using a Systems Biology Model." Blood 126, no. 23 (December 3, 2015): 2383. http://dx.doi.org/10.1182/blood.v126.23.2383.2383.

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Abstract To date numerous datasets of gene expression and epigenetic profiles for mouse and human hematopoietic cells have been generated. While individual data sets for a particular cell type have been correlated, no approach exists to harness all expression and epigenetic profiles for the different types of hematopoietic cells. Our goal is to develop a systems biology platform to compare epigenetic profiles of hematopoietic cells towards a better understanding of epigenetic mechanisms governing hematopoiesis. To provide the necessary foundation to support systematic studies of hematopoiesis, we have developed the Systems Biology Repository (SBR, http://sbrblood.nhgri.nih.gov), a data "ranch" for organizing and analyzing transcriptome and epigenome data cells throughout differentiation. To populate SBR, we extracted, curated, annotated, and integrated all human and mouse hematopoietic datasets available through the Encyclopedia of DNA Elements (ENCODE), the Gene Expression Omnibus (GEO) and the Short Read Repository (SRR). These include genome-wide profiles of DNA methylation, histone methylation and acetylation, transcription factor occupancy (ChIPSeq), chromatin accessibility (DNaseISeq, ATACSeq, FAIRESeq), and coding as well as non-coding transcriptional profiles (RNASeq). To demonstrate the utility of SBR, we conducted three different analyses. The first was a vertical study of HistoneSeq (H3K4me1, H3K4me2, H3K4me3, and H3K27ac), DNA methylation and RNASeq profiles during mouse erythroid differentiation. We found a global decrease in DNA methylation from hematopoietic stem and progenitor cells (HSC) through common myeloid progenitors (CMP), erythroid progenitor cells (MEP) and erythroblasts (ERY; 92936 peaks in HSC to 14422 in ERY). The number of expressed genes (using a tags per million cutoff of 10) increased in erythroid progenitors (8901 in HSC to 10778 in CMP and 10670 in MEP) before decreasing in ERY (8654). 62% of histone marks delineating active enhancers (H3K27ac, H3K4me1) are present in both HSC and ERY, while 48% arise de novo during differentiation. In contrast, only 16% of active promoter specific histone marks (H3K4me2, H3K4me3) are present in both HSC and ERY. For a horizontal analysis we compared the DNA methylation, RNASeq, histone modification (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) and transcription factor binding (GATA1 and NFE2) profiles of erythroblasts (ERY) and megakaryocytes (MEG). We found a similar relationship between gene expression and the histone and DNA methylation profiles in each cell type but differences between expression and in transcription factor occupancy. DNA methylation and H3K4me3 was enriched in the gene body of expressed genes (>36%) for both ERY (p ≤ 0.001) and MEG (p ≤ 0.01). In contrast DNA methylation was enriched in the upstream and downstream regions of non-coding RNA genes (p ≤ 0.001). Transcription factor occupancy was cell type specific: 79% of GATA1 sites are in ERY and 72% of NFE2 sites are in MEG. In erythroblasts, DNA methylation and GATA1 binding in the gene body are associated with gene silencing (4 fold difference, p ≤ 0.001), while in megakaryocytes, DNA methylation and NFE2 binding in the gene body are associated with gene activation (8 fold difference, p ≤ 0.001). We used the Mouse Genome Informatics homology map data to perform a cross-species comparison of the expression profiles of mouse and human multipotent progenitors (MPP), proerythroblasts and orthochromatic erythroblasts. We found a total of 5247 genes expressed at significantly different levels (p ≤ 0.001) between human and mouse MPP, while only 2010 genes were expressed at significantly similar levels (p ≤ 0.001). At the proerythroblast and orthochromatic erythroblast stages 7696 genes and 6571 genes were expressed at significantly different levels (p ≤ 0.001) between human and mouse respectively, while 2024 and 2560 genes were expressed at significantly similar levels (p ≤ 0.001). These data are consistent with previous studies showing differences in the transcriptional profiles of mouse and human hematopoietic cells. In summary, SBR provides a foundation to model the genetic and epigenetic landscape in both the mouse and human hematopoietic system, and enables functional correlations to be made between the species. As SBR is expanded to include data from patient cells, it will be possible to model epigenetic changes associated with disease. Disclosures No relevant conflicts of interest to declare.

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Fiskus, Warren, Rekha Rao, Ramesh Balusu, Jianguo Tao, Eduardo M. Sotomayor, Peter Atadja, and Kapil N. Bhalla. "Efficacy of Combined Epigenetic Targeting of Histone Methyltransferase EZH2 and Histone deacetylases Against Human Mantle Cell Lymphoma Cells." Blood 116, no. 21 (November 19, 2010): 2488. http://dx.doi.org/10.1182/blood.v116.21.2488.2488.

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Abstract Abstract 2488 Lysine specific histone methylation and deacetylation are chromatin modifications that, along with DNA methylation, are involved in the epigenetic silencing of tumor suppressor genes (TSGs). This silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity, which induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive histone modification mediating gene repression. The PRC1 components include BMI1, MEL18, RING1 and RING2, and it serves to further compact the chromatin at PRC2 target genes. The RING1 and RING2 proteins are responsible for the ubiquitylation of K119 on H2A. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharmaceutical Corp) depletes PRC2 complex proteins EZH2 and SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells (Blood 2009; 114: 2733–43). In the present studies we determined the effects of DZNep and/or PS on the expression of PRC1 and PRC2 proteins in human Mantle Cell Lymphoma (MCL) cells. Treatment with DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3, while inducing K27 acetylation on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2 in the cultured MCL cells. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and augmentation of H3K4Me3 chromatin marks. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in the MCL cells, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1 and MO2058, as well as of primary MCL cells derived from 3 patients with MCL (combination indices <1.0). Taken together these findings indicate that by targeted depletion of the PRC2 and PRC1 components and associated chromatin and other protein modifications (hsp90 acetylation), co-treatment with DZNep and PS represents a superior therapy of human MCL cells. These studies also support the in vivo testing of combined epigenetic therapies involving agents that target deregulated epigenetic mechanisms, e.g., histone deacetylases, methyl transferases and demethylases, as well as target DNMTs in the therapy of MCL. Disclosures: Atadja: Novartis: Employment.

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Gambacorta, Valentina, Daniela Gnani, Laura Zito, Stefano Beretta, Lucia Zanotti, Oliveira Giacomo, Davide Cittaro, et al. "Integrated Epigenetic Profiling Identifies EZH2 As a Therapeutic Target to Re-Establish Immune Recognition of Leukemia Relapses with Loss of HLA Class II Expression." Blood 134, Supplement_1 (November 13, 2019): 514. http://dx.doi.org/10.1182/blood-2019-127395.

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Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

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Szpurka, Hadrian, Anna M. Jankowska, Bartlomiej Przychodzen, Zhenbo Hu, Yogen Saunthararajah, Michael A. McDevitt, and Jaroslaw P. Maciejewski. "UTX Mutations and Epigenetic Changes In MDS/MPN and Related Myeloid Malignancies." Blood 116, no. 21 (November 19, 2010): 121. http://dx.doi.org/10.1182/blood.v116.21.121.121.

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Abstract Abstract 121 Balanced and unbalanced chromosomal lesions and genetic mutations are hallmarks of myeloid malignancies. In addition, aberrant methylation of CpG islands, leading to epigenetic silencing appears to play a significant role in tumor suppressor gene inactivation and malignant progression. While various mechanisms of chromosomal instability have been identified, the pathogenesis of epigenetic instability remains unexplored. ASXL1, EZH2 and TET2 mutations, found in myeloid disorders provide a potential link between genetic and epigenetic events. UTX is a histone H3K27 demethylase belonging to the polycomb group of proteins. Methylation at lysine 27 correlates with gene silencing and repression. A recent mutational screen of cancer cell lines has identified mutations in UTX in AML THP-1 cells. We screened a series of myeloid disorders for UTX gene mutations and identified an index case in a CMML patient with a somatic UTX missense mutation. This sample demonstrated × chromosome copy neutral loss of heterozygosity (CN-LOH) that included UTX on a whole genome SNP-A array. UTX was then sequenced in 49 additional patients with MDS/MPN and 24 with secondary AML. Two of the MDS/MPN (2/49, 4%) and 3 of the sAML patients (3/24; 12%) showed UTX mutation. Three previously unreported missense polymorphisms were noted (present in paired CD3+ cells and detected with a frequency of <1/400 of controls). No mutations were found in MDS (N=15). The identified lesions were not associated with loss of sex chromosomes. Both homozygous (UPD) and heterozygous UTX mutations were identified and included frame shift or stop codons, as well as missense mutations. Inactivating or hypomorphic mutations may result in similar functional consequences with decreased expression of UTX. We quantitated UTX mRNA abundance in 15 hematopoietic cell lines using TaqMan PCR, and THP1 cells (UTX null) showed the lowest UTX mRNA expression. In primary patient samples, decreased UTX mRNA levels were found in the bone marrow of MDS/MPN patients (N=21) as compared to controls. To examine UTX expression in normal hematopoietic differentiation, CD34+ cells, along with myeloid CD33+ or monocytic CD14+ cell fractions (N=5) were tested and expressed easily quantifiable UTX transcripts, while the highest UTX expression was found in Gly-A+ erythroid precursors. UTX mutation and dysfunction might be predicted to lead to altered histone repression marks and alterations in epigenetic regulation. Consistent with this hypothesis, we tested for H3K27-Me3 methylation by ELISA and observed a 45% methylation increase in a UTX mutant patient sample. To further investigate the function of UTX, consequences and treatment implications of the corresponding mutations we transduced THP-1 cells with a lentiviral vector containing the UTX cDNA (UTX+) or an empty vector. Following clone selection, the resultant UTX+ cell line showed 1000-fold UTX mRNA increase and distinct protein overexpression by western blot (WB), as compared to the baseline UTX null cells. Proliferation kinetics up to 120h indicated that overexpression of UTX results in increased cell proliferation by 20% compared to controls. Treatment with decitabine (120h; 0.5μ M) resulted in a differential effect on UTX+ cells: proliferation increased 4.6-fold vs. 1.3-fold in UTX null cells. Based on the hypothesis that mutation in UTX may lead to epigenetic instability and accumulation of aberrant methylated CpG sites, we further investigated epigenetic changes in UTX+ and UTX null cells. WB showed comparable levels of global H3K27-Me3 and H3 acetylation levels in UTX+ and UTX null cells, and only modest decreases (9%) of trimethylation in a more precise ELISA assay. Similarly, when methylation array (14K genes; 27K CpG sites) was used to assess the effects of UTX overexpression, global methylation levels expressed by averaged β-values did not significantly differ. However, 153 specific CpG sites were found to be differentially methylated on the array: with 68 genes hypo- and 85 hypermethylated. In particular SSRP1, NCOR2 and DIRAS3 genes showed hypomethylated promoters in the UTX+ cells, suggesting that UTX is involved in gene specific demethylation. In sum, our results suggest that UTX gene may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutation may compromise this function, thereby promoting repression of tumor suppressor genes. Disclosures: No relevant conflicts of interest to declare.

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Burda, Pavel, Nikola Curik, Nina Dusilkova, Giorgio L. Papadopoulos, John Strouboulis, Anna T. Jonasova, and Tomas Stopka. "Erythroid Transcription Factor GATA-1 Binds and Represses PU.1 Gene – Candidate Mechanism Of Epigenetic Repression Of PU.1 and Inefficient Erythropoiesis In MDS." Blood 122, no. 21 (November 15, 2013): 1558. http://dx.doi.org/10.1182/blood.v122.21.1558.1558.

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Abstract Introduction Myelodysplastic syndrome (MDS) is often manifested by anemia due to ineffective erythropoiesis. Upon transformation to MDS/AML the uniform population of leukemic blasts overgrow dysplastic bone marrow. Hematopoiesis is regulated by transcription factors GATA-1 and PU.1 that interact and mutually inhibit each other in progenitor cells to guide multilineage commitment and subsequent lineage differentiation. Expression of PU.1 is controlled by several transcription factors including PU.1 itself at distal URE enhancer. It has been well established that underexpression of PU.1 in progenitor cells leads to AML (Rosenbauer F et al. 2004). In addition, co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents induction of differentiation programs regulated by these transcription factors. In our laboratory, we recently observed that MDS/AML erythroblasts display repressive histone modifications and methylation status of PU.1 gene that respond to 5-azacitidine leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000) however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. Hypothesis GATA-1 inhibits PU.1 levels directly and modulates its transcriptional outcome in early erythroblasts. We also hypothesize that GATA-1-mediated repression of PU.1 transcription is delayed and this may play a role in ineffective erythropoiesis. Material and Methods Cell lines: MDS-derived OCI-M2 EL and other two human ELs (HEL, K562) and one murine EL (MEL); all co-expressing GATA-1 and PU.1. Patients: MDS patients (N=5) with rather advanced disease; MDS/AML (4) and RAEBI (1). Four received AZA; response: PR (2), SD (2) with HI. Median OS>24 Mo. For chromatin immunoprecipitation (ChIP) analysis either cell lines or CD19/CD3-depleted bone marrow cells were used. Results Direct association of GATA-1 with PU.1 gene was demonstrated in all three human ELs using ChIP. Occupancy of GATA-1 was detected upstream the PU.1 promoter and distally at GATA-1 binding sites or at PU.1 binding sites together with PU.1. Comparable data documenting occupancy of GATA-1 at PU.1 gene were observed also in MEL cells and in normal murine fetal erythroblasts using ChIP-sequencing. To test how GATA-1 regulates PU.1 expression we overexpressed GATA-1 in erythroblasts and tested expression of PU.1, histone H3 modification (near GATA-1 occupancy) and cell growth. We found that GATA-1 inhibited PU.1 expression, facilitated enrichment of repressive modifications at PU.1 gene (H3K9Me, H3K27Me) while depleted activation modifications (H3K9Ac, H3K4Me), and also inhibited cell growth. Next, we tested effects of GATA-1 knockdown using siRNA. Indeed, inhibition of GATA-1 expression in erythroblasts leads to increase in PU.1 level as well as of its targets (CEBPA, MAC1). Using Luciferase assay we confirmed that both endogenously produced PU.1 and GATA-1 are capable to stimulate exogenously inserted reporters. Next, we compared chromatin structure of PU.1 gene between data from ELs, normal controls and high risk MDS. Our data revealed that PU.1 gene in MDS is enriched with repressive modifications (H3K9Me, H3K27Me) while depleted with activation modifications (H3K9Ac, H3K4Me) suggesting defects in dynamic regulation of PU.1 expression in MDS. Conclusion Our data from ELs provide a) evidence of GATA-1-mediated repression of PU.1 gene in erythroblasts and that b) manipulation of GATA-1 affected PU.1 level in opposite direction. In high risk MDS, the chromatin structure of PU.1 gene displays accumulation of repressive epigenetic marks that are responsive to AZA. We think that during early erythroid differentiation GATA-1 binds and represses PU.1 gene, however this is not fully completed in MDS and therefore erythroid differentiation is not efficient. Grants: P301/12/P380, P305/12/1033, NT14174-3/2013, UNCE204021, FR-TI2/509, SVV-2013-266509, PRVOUK-P24/LF1/3 Disclosures: No relevant conflicts of interest to declare.

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Payton, Jacqueline E., Olivia I. Koues, Rodney Kowalewski, Jennifer A. Schmidt, Li-Wei Chang, Amanda Cashen, Nancy L. Bartlett, and Eugene M. Oltz. "Defining the Malignant Epigenome in Non-Hodgkin Lymphoma." Blood 120, no. 21 (November 16, 2012): 524. http://dx.doi.org/10.1182/blood.v120.21.524.524.

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Abstract Abstract 524 Understanding epigenetic mechanisms of gene regulation will provide an unprecedented opportunity for therapeutic intervention in cancer because, unlike genetic lesions, pathogenic changes to the epigenome are reversible. Non-Hodgkin Lymphoma (NHL), which strikes 70,000 Americans annually, is characterized by deregulated expression of large gene cohorts that mediate unchecked cell growth, the molecular basis of which remains poorly understood. Recently, recurrent somatic mutations were identified in chromatin modifier genes (EZH2, MLL2, EP300) in ∼30% of NHL, suggesting that epigenetic dysregulation may be a common mechanism for widespread gene expression changes. Indeed, previous studies have demonstrated evidence of aberrant DNA methylation in lymphoma. Moreover, NHL-specific regulatory elements are attractive candidates for therapeutic targeting, because reversal of their aberrant epigenetic profile would restore normal gene expression, while leaving normal cells intact. In this study, we performed integrative epigenomic, genomic, and transcriptomic profiling of purified CD19+ B cells from excisional lymph node biopsies and peripheral blood (PB, when available and free of circulating lymphoma) in 84 patients with NHL (Diffuse Large B cell lymphoma (DLBCL) N=34, Follicular Lymphoma (FL) N=38, Marginal Zone/Mantle Cell/Chronic Lymphocytic Leukemia (MZL/MCL/CLL) N=12), 20 patients undergoing elective tonsillectomy (tonsil B cells), and 40 healthy volunteers (PB). To enrich for key changes in the NHL epigenome, crosslinked chromatin was subjected to formaldehyde-assisted isolation of regulatory elements (FAIRE), for highly active histone-depleted regions, and chromatin immunoprecipitation (ChIP) for several activating (H3K4me1, H3K27ac, H3K9/14ac), and repressive (H3K27me3) histone marks, followed by high throughput sequencing (FAIRE/ChIP-Seq). To identify genes regulated by putative NHL-specific regulatory elements, we profiled gene expression (mi/mRNA) by microarray and, in primary tumors, by RNA-Seq to detect novel isoforms, translocations, and mutations, such as those in epigenetic modifier genes. In order to analyze this complex dataset, we created a global, integrative algorithm to evaluate the regulatory potential of a given epigenetic element, or set of elements, based on a step-wise approach. All regulatory elements were ranked according to several parameters, including distance to transcription start sites (TSS), gene expression level, chromatin state, distance to CTCF sites, genomic copy number, degree of sequence conservation, association with mutations in epigenetic modifiers, and most importantly, NHL-specificity and recurrence (number of samples). We first established the NHL-specificity of each regulatory element by comparison to matched PBB, if available, or to healthy donor B cells, identifying thousands of regulatory elements that are differentially enriched (“gained” or “lost”) in at least one primary NHL sample. We further assessed the likely role of each element by its genomic, epigenomic, and transcriptomic contexts. Gained elements were significantly associated with higher levels of gene expression in NHL compared to normal B cells (p<0.0001); and conversely, lost elements were significantly associated with decreased expression levels (p<0.0035). Subsequent to this global analysis, we ranked the NHL-specific elements as described above and identified a set of prioritized signatures, comprising a few dozen to hundreds of elements, depending on the type (gained/lost), chromatin state, and recurrence. As predicted, some of these signatures were associated with oncogenes, such as TP63, BCL2, and BCL6; however, others are intergenic and may be cis-elements coordinating expression of distal genes. Intriguingly, subsets of these chromatin signatures are specific for distinct NHL subtypes or grades. In summary, our findings establish a foundational dataset for surveying the epigenomic landscape of NHL, while identifying key chromatin signatures for the development of targeted epigenetic therapies. Disclosures: No relevant conflicts of interest to declare.

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Woo, Andrew J., Jonghwan Kim, Jian Xu, Hui Huang, and Alan Cantor. "Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development." Blood 116, no. 21 (November 19, 2010): 2067. http://dx.doi.org/10.1182/blood.v116.21.2067.2067.

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Abstract Abstract 2067 The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells. Disclosures: No relevant conflicts of interest to declare.

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Karkhanis, Vrajesh, Olivier Elemento, Maurizio Di Liberto, Selina Chen-Kiang, Said Sif, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Directly Targets and Epigenetically Silences microRNAs miR33b and miR96 to Support Constitutive Cyclin D1 Activity in Non-Hodgkin’s Lymphoma." Blood 124, no. 21 (December 6, 2014): 60. http://dx.doi.org/10.1182/blood.v124.21.60.60.

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Abstract Epigenetic regulation mediated by arginine-specific methyltransferases enzymes play a central role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 has been associated with increased cell proliferation and survival. We have previously demonstrated that PRMT5 is over expressed in mantle cell lymphoma (MCL) and supports constitutive CYCLIN D1/CDK4/6 activity leading to inactivation of the RB/E2F pathway. PRMT5 is also a driver of PRC2 epigenetic activity and promotes activation of b-CATENIN target genes MYC and SURVIVIN (Chung et al ASH, 2014). While PRMT5 has been characterized as a transcriptional silencer, few genes have been identified as direct targets. In this study, we have taken advantage of the next generation sequencing (NGS) platform and integrated high throughput Illumina High-seq ChIP sequencing with whole transcriptome analysis to shed light on the global epigenetic role played by PRMT5 in B-cell lymphoma. We have conducted ChIP-sequencing analysis to map genome-wide recruitment of the PRMT5-specific epigenetic mark H3Me2R8 in three patient-derived non-Hodgkin’s lymphoma (NHL) cell lines (pre germinal center (GC) Jeko, GC Pfeiffer, and post GC SUDHL2) as well as control Normal B-cells. Our results indicate that PRMT5 targets as many as 8500 genes in each lymphoma cell line and there are approximately 4600 genes that are common targets between the three different lymphoma cell lines. We next performed genome-wide mRNA sequencing of each lymphoma cell line after treating with a lentivirus expressing PRMT5 shRNA (or GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5, or DMSO vehicle control). Evaluation of direct microRNA (miR) targets common to all three NHL cell lines showed that the PRMT5-driven epigenetic mark H3Me2R8 was enriched on the promoters of miR33b, miR96 and miR506, three miRs predicted to bind the 3’ untranslated region (UTR) of CYCLIN D1. RNA-Seq and ChIP experiments from NHL samples treated with control or PRMT5 inhibition suggested that PRMT5 epigenetically silenced all three microRNAs and validation studies using real-time RT-PCR demonstrated that PRMT5 knock down/inhibition led to transcriptional derepression of miR33b and miR96 in all three cell lines and primary patient mantle cell lymphoma samples. Inhibition of PRMT5 led to loss of recruitment of epigenetic co-repressor complexes containing PRMT5 and HDAC3 and gain of p65 and enrichment of hyperacetylated lysine epigenetic marks H4K8, H3K14 and H2BK12. Interestingly, suppression of both of these miRNAs was also found to augment PRMT5 translation and re-expression of miR33b and miR96 led to simultaneous down regulation of PRMT5 and CYCLIN D1. Using wild-type and mutant CYCLIN D1 3’ UTRs subcloned downstream of a CMV driven luciferase reporter, we show that the binding sites of miR33b and miR96 are critical for translational regulation by these miRNAs. Our studies link dysregulated PRMT5 expression, which is a common finding in aggressive lymphomas, to aberrant expression of miR33b and miR96 and highlights yet another mechanism by which this arginine methyltransferase can support the expression of CYCLIN D1 in transformed lymphoma cell lines and MCL clinical samples. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own miRNAs and those that regulate CYCLIN D1, and that its inhibition could offer a promising therapeutic strategy for lymphoma patients. Disclosures No relevant conflicts of interest to declare.

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Chung, Jihyun, Vrajesh Karkhanis, Sif Said, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Regulates WNT/β-Catenin Target Gene Expression in at Multiple Levels." Blood 128, no. 22 (December 2, 2016): 4106. http://dx.doi.org/10.1182/blood.v128.22.4106.4106.

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Abstract Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA. Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters. Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma. Disclosures Baiocchi: Essanex: Research Funding.

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Vaitkus, Kestis, Vinzon Ibanez, Maria Armila Ruiz, Ramasamy Jagadeeswaran, Yogenthiran Saunthararajah, James Douglas Engel, Joseph DeSimone, Angela Rivers, and Donald Lavelle. "The LSD1 Inhibitor RN-1 Increases γ-Globin Expression in Baboons By Targeting an Early Event Responsible for γ-Globin Repression." Blood 132, Supplement 1 (November 29, 2018): 1054. http://dx.doi.org/10.1182/blood-2018-99-113539.

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Abstract The HbF to HbA developmental globin switch is recapitulated during adult erythroid differentiation and involves the acquisition of repressive epigenetic marks at the γ-globin promoter catalyzed by "druggable" enzymes such as histone deacetylases (HDACs), DNA methyltransferase (DNMT1), LSD1 (KDM1A), and G9A (EHMT2) that are functional components of multiprotein co-repressors recruited to the γ-globin gene promoter by the trans-acting repressors TR2/TR4, BCL11A, and ZBTB7A. A logical approach to increase HbF that has been successfully pursued by our laboratory is to intervene with the epigenetic repression mechanism that executes the switch from HbF to HbA using pharmacological inhibitors of these enzymes. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates.Our laboratory has developed and utilized an in vivo baboon model for over thirty years to investigate globin gene regulation and the ability of pharmacological inhibitors of enzymes that catalyze repressive epigenetic modifications to increase HbF, generating results with DNMT inhibitors that were extended and confirmed in a number of clinical studies in patients with SCD and β-thalassemia. Following studies that identified LSD1 as an additional therapeutic target (Shi et al, Nat Med 19:291, 2013), we showed that the LSD1 inhibitor RN-1 dramatically increased HbF, F cells and F retics in baboons (Rivers et al, Haematol 101:698, 2015) and that these effects were sustained upon long-term treatment (>265d; Ibanez et al, Blood 129:260, 2017). ChIP analysis showed increased levels of Histone H3 di and tri-methyl K4 at the γ-globin gene, consistent with LSD1 inhibition. This current investigation seeks to identify the stage in the erythroid differentiation pathway targeted by LSD1 inhibitors to increase HbF and also to characterize additional effects of these drugs on erythroid differentiation that potentially impact mechanism of action. Subpopulations of bone marrow (BM) cells highly enriched in BFUe (CD105+CD34+CD117+bRBC-), CFUe (CD105+CD34+CD117+bRBC+), proerythroblasts (CD105+CD34-CD117+bRBC+) were purified from RN-1 treated (0.25mg/kg/d; 3d) and untreated baboons by immunomagnetic column separation combined with FACS. RT-PCR analysis of γ-globin expression within purified BM erythroid subpopulations showed that relative levels of γ-globin mRNA (γ/γ+β) in untreated baboons were 4 fold higher in BFUe than in CFUe and 11 fold higher in BFUe than in proerythroblasts consistent with repression of the γ-globin gene during the BFUe to CFUe transition. RN-1 treatment increased relative γ-globin expression (γ/γ+β) 4 fold in BFUe, 25 fold in CFUe, and >100 fold in proerythroblasts compared to untreated controls and thus partially prevented repression of the γ-globin expression at early stages of differentiation. Additional effects of the LSD1 inhibitor RN-1 on erythroid differentiation were measured by flow cytometry in baboon BM aspirates following RN-1 administration. RN-1 treatment (0.25mg/kg/d; 3d) increased the proportion of CD105+CD117+bRBC+BM cells (proerythroblasts) (24.2% pre-treatment; 48.4% post-treatment; p<0.02) and decreased the proportion of CD105+CD117-bRBC+ terminal erythroid BM cells (51.1% pre-treatment; 29.1% post-treatment; p<0.02). RNAseq analysis of CD105+CD34-CD117+bRBC+ FACS-purified BM cells (proerythroblasts) of untreated control (n=2) and RN-1 treated baboons (0.25mg/kg/d; 3d; n=4; 2 treated for 3d; 2 treated >265d) identified a common set of 41 genes whose expression was increased while the expression of 6 genes was decreased. RT-PCR assays confirmed increased expression of γ-globin, GATA-2, GFi-1B, DAP, IFI6, and Lyn in proerythroblasts of RN-1-treated baboons. We conclude that the LSD1 inhibitor RN-1 administered to baboons 1) restrains erythroid differentiation by modulating the expression of genes such as GATA-1 and GFi-1B that regulate erythroid differentiation, and 2) targets an early event responsible for γ-globin repression to increase γ-globin expression. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Lavelle:Global Blood therapeutics: Research Funding.

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Bapat, Sharmila A., Victor Jin, Nicholas Berry, Curt Balch, Neeti Sharma, Nawneet Kurrey, Shu Zhang, et al. "Multivalent epigenetic marks confer microenvironment-responsive epigenetic plasticity to ovarian cancer cells." Epigenetics 5, no. 8 (November 2010): 716–29. http://dx.doi.org/10.4161/epi.5.8.13014.

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Hu, Xin, Xingguo Li, River Ybarra, Kristell Valverde, Xueqi Fu, Constance Noguchi, Yi Qiu, and Suming Huang. "LSD1-Mediated Epigenetic Modification Is Important for TAL1 Function." Blood 112, no. 11 (November 16, 2008): 4757. http://dx.doi.org/10.1182/blood.v112.11.4757.4757.

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Abstract TAL1/SCL is critical for normal and abnormal hematopoiesis by regulating hematopoietic stem/progenitor cell growth and differentiation. However, it is still unclear how its transcriptional activities are controlled during hematopoiesis. Here, we undertook the biochemical isolation of TAL1-associated protein complexes in erythroleukemia cells and showed that TAL1 interacts with histone demethylase LSD1 complexes containing LSD1, CoREST, HDAC1 and HDAC2. Interestingly, although TAL1 specifically colocalizes with LSD1 at the target gene promoter p4.2 in undifferentiated MEL cells, the recruitment of LSD1 is decreased at the p4.2 promoter upon induced MEL differentiation indicating that LSD1 may differentially regulate TAL1 target genes during differentiation. The siRNA-mediated knockdown of LSD1 in MEL and ES cells resulted in the derepression of p4.2 by increasing dimeH3K4 at their promoter region, respectively. Finally, we demonstrated that TAL1-associated LSD1 complexes, H3K4 demethylase, and histone deacetylase activities are coordinately regulated during erythroid cell differentiation. Thus, the data suggest that LSD1 mediated epigenetic modification may affect hematopoiesis and leukemogenesis through its association with the lineage-specific transcription factor TAL1.

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Stanworth, S. J., N. A. Roberts, J. A. Sharpe, J. A. Sloane-Stanley, and W. G. Wood. "Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids." Molecular and Cellular Biology 15, no. 8 (August 1995): 3969–78. http://dx.doi.org/10.1128/mcb.15.8.3969.

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Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.

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Kint, Sam, Wim Van Criekinge, Linos Vandekerckhove, Winnok H. De Vos, Karol Bomsztyk, Diane S. Krause, and Oleg Denisenko. "Single cell epigenetic visualization assay." Nucleic Acids Research 49, no. 8 (January 28, 2021): e43-e43. http://dx.doi.org/10.1093/nar/gkab009.

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Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.

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Wibowo, Anjar, Claude Becker, Julius Durr, Jonathan Price, Stijn Spaepen, Sally Hilton, Hadi Putra, et al. "Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation." Proceedings of the National Academy of Sciences 115, no. 39 (September 10, 2018): E9145—E9152. http://dx.doi.org/10.1073/pnas.1805371115.

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Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells. This unique developmental plasticity is commonly observed in nature, where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs. While organ-specific epigenetic marks are not passed on during sexual reproduction, the fate of epigenetic marks during asexual reproduction and the implications for clonal progeny remain unclear. Here we report that organ-specific epigenetic imprints in Arabidopsis thaliana can be partially maintained during asexual propagation from somatic cells in which a zygotic program is artificially induced. The altered marks are inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the identity of the founder tissue. Consequently, clonal plants display distinct interactions with beneficial and pathogenic microorganisms. Our results demonstrate how novel phenotypic variation in plants can be unlocked through altered inheritance of epigenetic marks upon asexual propagation.

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Ramanouskaya, Tatsiana V., Anastasiya V. Kviatko, and Vasily V. Grinev. "Epigenetic marks on the chromatin are associated with RNA splicing in human leukemia cells." Journal of the Belarusian State University. Biology, no. 2 (July 18, 2019): 70–81. http://dx.doi.org/10.33581/2521-1722-2019-2-70-81.

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In this work we estimated associations between distribution patterns of several epigenetic marks and splicing events on the level of full genome and transcriptome in the cells of two leukemic cell lines containing two different reciprocal chromosome translocations. Significant difference in distribution of epigenetic marks was found, contributing to more opened or more closed chromatin in loci of donor vs acceptor and canonical vs alternative splice sites in expressing genes. Marks of the opened chromatin are significantly more often present in the genomic regions with alternative splicing events than in regions with canonical splicing, while for the mark of the histone 3 trimethylation at lysine 36, the opposite trend is observed. The obtained results reveal the presence of an additional, still very poorly studied layer in the regulation of alternative splicing in human cells.

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Negi, Sandeep S., Eric S. Schafer, Donald Small, and Patrick Brown. "Histone Profiling of Normal B-Precursors and Primary Pre-B Acute Lymphoblastic Leukemia Reveals Distinct Aberrant Histone Codes In MLL-Rearranged Vs. Wild Type MLL Leukemias That Correlate with Differential Expression of Key MLL Target Genes." Blood 116, no. 21 (November 19, 2010): 2503. http://dx.doi.org/10.1182/blood.v116.21.2503.2503.

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Abstract 2503 Epigenetic regulation of gene transcription is mediated both by methylation of DNA CpG islands and the local configuration of chromatin, which is dynamically regulated by post-translational modifications, or “marks”, of key lysines (K) of histones (especially H3). Some marks are associated with transcriptional repression [trimethylation (me3) of K9 and K27], and some with activation [me3 of K4, dimethylation (me2) of K79 and acetylation (Ac) of K9 and K14]. The MLL gene encodes a protein that functions as a master regulator of target gene expression by methylating H3K4 via its SET domain, and by interacting with other proteins with histone modifying properties. MLL is frequently rearranged (MLL-r) by translocations in acute leukemias, which exhibit a distinct global gene expression pattern. Many MLL-r partner genes form complexes that can methylate H3K79. Thus, histone modifications may be central to the function of both wild type (MLL-wt) and MLL-r. We hypothesized that aberrant histone coding of target genes contributes to MLL-r leukemogenesis. We characterized the histone code associated with the promoters of selected genes in n=5 MLL-r pre-B ALL samples (MLL-AF4 or MLL-ENL), n=4 MLL-wt pre-B ALL samples (TEL-AML1 or hyperdiploid) and normal control B-precursors (CD19+ cord blood cells). We selected 9 genes differentially overexpressed in MLL-r leukemia (HOXA7, HOXA9, MEIS1, FLT3, CCNA1, ZC3H12C, ATP8B4, C20orf103, and PROM1), and 3 control genes that are not MLL targets (HOXA1, HOXC8, LTF). We performed ChIP with antibodies specific for key H3 modifications (K4me3, K9me3, K9/14Ac, K27me3 and K79me2), followed by qPCR for the selected genes. Expression was measured by RT/qPCR. All 9 MLL target genes were significantly overexpressed in the MLL-r cohort, and this was associated with a specific “activating” histone code at the genes' promoters (fig 1 – MEIS1, e.g.). The opposite “repressive” code was found in the MLL-wt cohort, and in the MLL-r cohort at the promoters of the control genes. Compared to both sets of leukemias, normal B-precursors exhibited a paucity of histone modifications for all genes. For most genes, a specific developmental pattern of alterations in the histone code and corresponding relative change in expression could be traced from the normal B-precursors to the leukemia cells. This pattern was strikingly different in MLL-r leukemias than in MLL-wt leukemias, suggesting that the acquisition of MLL-r by normal B-precursors causes altered gene expression patterns via changes in the histone code. For most genes, normal B-precursors exhibit both the activating K4me3 mark and the repressive K27me3 mark, and express low but detectable levels of RNA. In MLL-r leukemias, upregulation of genes is associated with an increase in K4me3, loss of K27me3, and gain of K9/14Ac and/or K79me2. In MLL-wt leukemias, silencing of genes is associated with loss of K4me3 and gain of K9me3. To study the direct effects of MLL-wt and MLL-r on the histone code, we used 2 rounds of siRNA over 48 hours to knock down MLL-AF4 only, MLL-wt only or both in the RS4;11 cell line (MLL-AF4+ B-precursor ALL), then performed RT/qPCR and ChIP/qPCR. We achieved at least 60% knock down of MLL-AF4 and/or MLL-wt. Knock down of MLL-wt, with or without concomitant knock down of MLL-AF4, did not diminish the K4me3 mark for any genes, suggesting that MLL's SET domain is not required to maintain K4 methylation. While knock down of MLL-AF4 or MLL-wt alone did not diminish K79 methylation, knock down of both completely removed the K79me2 mark from all genes, suggesting that expression of either MLL-wt or MLL-AF4 is absolutely required for H3K79 methyltransferase activity. Two genes (HOXA7 and PROM1) demonstrated evidence of direct transcriptional regulation by MLL-AF4, since their expression decreased markedly after knock down of MLL-AF4 alone or with MLL-wt, but not with MLL-wt alone. In summary, primary MLL-r pre-B ALLs exhibit a distinct activating histone code at key overexpressed target genes when compared to MLL-wt pre-B ALLs and normal B-precursors. A causative role for MLL fusion proteins is suggested by the distinct pattern of histone code progression from normal B-precursors to MLL-r leukemias. Furthermore, knock down experiments provide direct evidence that some of the observed histone modifications in MLL-r leukemia, particularly H3K79 methylation, are directly downstream of wild type and mutant MLL. Disclosures: No relevant conflicts of interest to declare.

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Patra, Samir Kumar, Moonmoon Deb, and Aditi Patra. "Molecular marks for epigenetic identification of developmental and cancer stem cells." Clinical Epigenetics 2, no. 1 (December 17, 2010): 27–53. http://dx.doi.org/10.1007/s13148-010-0016-0.

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Uller, Tobias, Sinead English, and Ido Pen. "When is incomplete epigenetic resetting in germ cells favoured by natural selection?" Proceedings of the Royal Society B: Biological Sciences 282, no. 1811 (July 22, 2015): 20150682. http://dx.doi.org/10.1098/rspb.2015.0682.

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Resetting of epigenetic marks, such as DNA methylation, in germ cells or early embryos is not always complete. Epigenetic states may therefore persist, decay or accumulate across generations. In spite of mounting empirical evidence for incomplete resetting, it is currently poorly understood whether it simply reflects stochastic noise or plays an adaptive role in phenotype determination. Here, we use a simple model to show that incomplete resetting can be adaptive in heterogeneous environments. Transmission of acquired epigenetic states prevents mismatched phenotypes when the environment changes infrequently relative to generation time and when maternal and environmental cues are unreliable. We discuss how these results may help to interpret the emerging data on transgenerational epigenetic inheritance in plants and animals.

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Cheng, Xiaodong, Hideharu Hashimoto, Yusuf Olatunde Olanrewaju, Samuel Hong, and Xing Zhang. "Generation, Recognition, and Erasure of 5-methylcytosine and its Oxidative Derivatives." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C297. http://dx.doi.org/10.1107/s2053273314097022.

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During the development, mammalian germ line cells and brains undergo a series of cellular and molecular events that lead to the erasure and re-establishment of epigenetic programs. It is possible that the active erasure and the re-establishment of 5-methylcytosine (5mC) marks during germ line differentiation and brain development from fetus to young adult involve dynamic changes of 5mC into oxidative marks, and that these modified cytosine residues in DNA are recognized by specific protein readers with distinct roles in the maintenance of epigenetic memory. Here we report our on-going biochemical and structural analyses of generation, recognition and erasure of these marks.

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Harandi-Zadeh, Sadaf, Cayla Boycott, Megan Beetch, Tony Yang, Benjamin J. E. Martin, Kevin Ren, Anna Kwasniak, et al. "Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells." Antioxidants 10, no. 8 (July 30, 2021): 1232. http://dx.doi.org/10.3390/antiox10081232.

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Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 μM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

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Kamada, Rui, Wenjing Yang, Yubo Zhang, Mira C. Patel, Yanqin Yang, Ryota Ouda, Anup Dey, et al. "Interferon stimulation creates chromatin marks and establishes transcriptional memory." Proceedings of the National Academy of Sciences 115, no. 39 (September 10, 2018): E9162—E9171. http://dx.doi.org/10.1073/pnas.1720930115.

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Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNβ stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNβ-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

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Ohguchi, Hiroto, Teru Hideshima, Manoj Bhasin, Gullu Gorgun, Loredana Santo, Michele Cea, Naoya Mimura, et al. "The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival." Blood 126, no. 23 (December 3, 2015): 3633. http://dx.doi.org/10.1182/blood.v126.23.3633.3633.

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Abstract Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase. KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM. KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A. We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells. shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells. KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow. In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. Disclosures Raje: Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

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Capparelli, Rosanna, and Domenico Iannelli. "Role of Epigenetics in Type 2 Diabetes and Obesity." Biomedicines 9, no. 8 (August 8, 2021): 977. http://dx.doi.org/10.3390/biomedicines9080977.

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Epigenetic marks the genome by DNA methylation, histone modification or non-coding RNAs. Epigenetic marks instruct cells to respond reversibly to environmental cues and keep the specific gene expression stable throughout life. In this review, we concentrate on DNA methylation, the mechanism often associated with transgenerational persistence and for this reason frequently used in the clinic. A large study that included data from 10,000 blood samples detected 187 methylated sites associated with body mass index (BMI). The same study demonstrates that altered methylation results from obesity (OB). In another study the combined genetic and epigenetic analysis allowed us to understand the mechanism associating hepatic insulin resistance and non-alcoholic disease in Type 2 Diabetes (T2D) patients. The study underlines the therapeutic potential of epigenetic studies. We also account for seemingly contradictory results associated with epigenetics.

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Sloan, Shelby, Fiona Brown, JI Hyun Chung, Alexander Prouty, Esther Wheeler, Bonnie K. Harrington, Eric Brooks, et al. "Targeting PRMT5 to Circumvent Acquired Ibrutinib Resistance in Mantle Cell Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 4065. http://dx.doi.org/10.1182/blood-2019-128998.

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Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by genetic dysregulation of cyclin D1 and activation of signaling pathways driving uncontrolled MCL cell proliferation and survival. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK), a downstream target of the B-cell receptor (BCR) pathway. While ibrutinib exhibits significant single-agent therapeutic activity in patients with relapsed/refractory MCL, the vast majority of MCL patients on ibrutinib progress with aggressive disease and short survival (3-8 mo). Although ~80% of chronic lymphocytic leukemia patients with acquired ibrutinib resistance have mutations in BTK and PLCγ2, this is uncommon in MCL suggesting alternative mechanisms driving this resistant phenotype. Understanding drug-resistance mechanisms and developing effective therapies for ibrutinib resistant (IR) MCL are urgently needed. The major type II protein arginine methyltransferase enzyme, PRMT5, catalyzes symmetric dimethylation of arginine residues on histone tails (H3R8 and H4R3) and other proteins. PRMT5 regulates a vast array of biologic functions including RNA processing, DNA damage response, signal transduction, and gene expression. Amplified PRMT5 activity drives the expression and activity of key oncogenes (MYC, CYCLIND1, NOTCH1) while silencing expression and activity of tumor suppressors (ST7, RBL2, and p53). Our group has shown PRMT5 is overexpressed and dysregulated in MCL and strategies aimed at selectively targeting PRMT5 show anti-tumor activity in preclinical lymphoma models. Here we describe the development of a novel patient derived xenograft (PDX) of IR-MCL and explore PRMT5 inhibition as an alternative therapeutic option to circumvent IR. Peripheral blood mononuclear cells from a 75 yo male patient diagnosed with acquired classic IR-MCL were engrafted intravenously into NSG mice. After 5 passages, all mice engrafted with 107 MCL cells developed histologically confirmed MCL infiltrating kidney, lymph nodes, bone marrow, spleen and peripheral blood. Circulating human CD5+/CD19+ cells were detectable and quantifiable by flow cytometry by day 21 post-engraftment. Karyotype analysis confirmed the hallmark t(11;14)(q13;q32) of MCL while retaining nearly all cytogenetic abnormalities present in the patient's primary tumor including a deletion of chromosome 9, associated with deletion of MTAP, a therapeutic vulnerability for PRMT5-targeted therapy. Whole exome sequencing confirmed genomic stability with successive passages. Ex vivo cytotoxicity assays and protein pathway analysis further confirmed resistance to ibrutinib (IC50 >1 µM) with maintained hyper-phosphorylation of AKT (Ser473) and ERK (Thr202/Tyr204). Western blot analysis showed elevated levels of c-MYC, CYCLIND1, BCL2, and pERK. After validation of circulating disease at day 25 post engraftment, mice were treated with either a novel small molecule inhibitor of PRMT5 (PRT382, 10 mg/kg orally 4 days on 3 days off) or ibrutinib (75 mg/kg administered in drinking water, n=5 mice per treatment group). Treatment of this PDX model with PRT382 resulted in significantly decreased disease burden and improved median survival compared to control animals from 48 to 83 days, respectively (p=0.0045). We found no significant difference in survival (p= 0.6540) or circulating disease burden with ibrutinib therapy compared to control group. The full BTK occupancy of ibrutinib treated mice was validated using fluorescence resonance energy transfer-based assay. Ex vivo PDX MCL cells from PRT382-treated mice showed loss of symmetric dimethyl arginine with preservation of asymmetric dimethyl arginine levels, reduced H4(Sme2)R3 epigenetic marks, and elevated levels of BCL2, MYC, and pAKT/pERK. We developed a cell line (SEFA) allowing for in vitro mechanistic studies. We are currently investigating potential mechanisms responsible for circumventing IR-MCL by integrating genome-wide changes to chromatin accessibility and whole transcriptome analysis. This IR-MCL PDX mouse model serves as a useful tool to investigate mechanisms of drug resistance, provides a platform to explore novel pre-clinical therapeutic strategies to circumvent IR and demonstrates the therapeutic activity of PRMT5 targeted therapy in this aggressive disease. Disclosures Byrd: Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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Antony, J., F. Oback, R. Broadhurst, S. Cole, C. Graham, T. Jenuwein, L. Chamley, B. Oback, and G. Laible. "500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS." Reproduction, Fertility and Development 21, no. 9 (2009): 101. http://dx.doi.org/10.1071/srb09abs500.

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To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

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Zarakowska, Ewelina, Jolanta Czerwinska, Agnieszka Tupalska, Matt J. Yousefzadeh, Siobhán Q. Gregg, Claudette M. St Croix, Laura J. Niedernhofer, et al. "Oxidation Products of 5-Methylcytosine are Decreased in Senescent Cells and Tissues of Progeroid Mice." Journals of Gerontology: Series A 73, no. 8 (February 3, 2018): 1003–9. http://dx.doi.org/10.1093/gerona/gly012.

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Abstract 5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1−/− primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2′-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2′-deoxyuridine and 5-(hydroxymethyl)-2′-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2′-deoxycytidine and 5-formyl-2′-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2′-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.

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Robbez-Masson, Luisa, Christopher H. C. Tie, and Helen M. Rowe. "Cancer cells, on your histone marks, get SETDB1, silence retrotransposons, and go!" Journal of Cell Biology 216, no. 11 (October 24, 2017): 3429–31. http://dx.doi.org/10.1083/jcb.201710068.

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Cancer cells thrive on genetic and epigenetic changes that confer a selective advantage but also need strategies to avoid immune recognition. In this issue, Cuellar et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201612160) find that the histone methyltransferase SETDB1 enables acute myeloid leukemia cells to evade sensing of retrotransposons by innate immune receptors.

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Arnaud, Philippe. "Genomic imprinting in germ cells: imprints are under control." REPRODUCTION 140, no. 3 (September 2010): 411–23. http://dx.doi.org/10.1530/rep-10-0173.

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The cis-acting regulatory sequences of imprinted gene loci, called imprinting control regions (ICRs), acquire specific imprint marks in germ cells, including DNA methylation. These epigenetic imprints ensure that imprinted genes are expressed exclusively from either the paternal or the maternal allele in offspring. The last few years have witnessed a rapid increase in studies on how and when ICRs become marked by and subsequently maintain such epigenetic modifications. These novel findings are summarised in this review, which focuses on the germline acquisition of DNA methylation imprints and particularly on the combined role of primary sequence specificity, chromatin configuration, non-histone proteins and transcriptional events.

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de Miranda, Juliana Xavier, Fábia de Oliveira Andrade, Aline de Conti, Maria Lúcia Zaidan Dagli, Fernando Salvador Moreno, and Thomas Prates Ong. "Effects of selenium compounds on proliferation and epigenetic marks of breast cancer cells." Journal of Trace Elements in Medicine and Biology 28, no. 4 (October 2014): 486–91. http://dx.doi.org/10.1016/j.jtemb.2014.06.017.

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Fessele, Kristen L., and Fay Wright. "Primer in Genetics and Genomics, Article 6: Basics of Epigenetic Control." Biological Research For Nursing 20, no. 1 (November 23, 2017): 103–10. http://dx.doi.org/10.1177/1099800417742967.

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The epigenome is a collection of chemical compounds that attach to and overlay the DNA sequence to direct gene expression. Epigenetic marks do not alter DNA sequence but instead allow or silence gene activity and the subsequent production of proteins that guide the growth and development of an organism, direct and maintain cell identity, and allow for the production of primordial germ cells (PGCs; ova and spermatozoa). The three main epigenetic marks are (1) histone modification, (2) DNA methylation, and (3) noncoding RNA, and each works in a different way to regulate gene expression. This article reviews these concepts and discusses their role in normal functions such as X-chromosome inactivation, epigenetic reprogramming during embryonic development and PGC production, and the clinical example of the imprinting disorders Angelman and Prader–Willi syndromes.

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Rhie, Suhn K., Shannon Schreiner, Heather Witt, Chris Armoskus, Fides D. Lay, Adrian Camarena, Valeria N. Spitsyna, et al. "Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation." Science Advances 4, no. 12 (December 2018): eaav8550. http://dx.doi.org/10.1126/sciadv.aav8550.

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As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.

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Nesvick, Cody, Charles Day, Liang Zhang, Edward Hinchcliffe, and David Daniels. "DIPG-78. REVERTANCE OF THE H3K27M MUTATION RESCUES CHROMATIN MARKS NECESSARY FOR ONCOGENESIS IN DIFFUSE MIDLINE GLIOMA." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii302. http://dx.doi.org/10.1093/neuonc/noaa222.120.

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Abstract Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.

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Pruvost, Mathilde, and Sarah Moyon. "Oligodendroglial Epigenetics, from Lineage Specification to Activity-Dependent Myelination." Life 11, no. 1 (January 15, 2021): 62. http://dx.doi.org/10.3390/life11010062.

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Oligodendroglial cells are the myelinating cells of the central nervous system. While myelination is crucial to axonal activity and conduction, oligodendrocyte progenitor cells and oligodendrocytes have also been shown to be essential for neuronal support and metabolism. Thus, a tight regulation of oligodendroglial cell specification, proliferation, and myelination is required for correct neuronal connectivity and function. Here, we review the role of epigenetic modifications in oligodendroglial lineage cells. First, we briefly describe the epigenetic modalities of gene regulation, which are known to have a role in oligodendroglial cells. We then address how epigenetic enzymes and/or marks have been associated with oligodendrocyte progenitor specification, survival and proliferation, differentiation, and finally, myelination. We finally mention how environmental cues, in particular, neuronal signals, are translated into epigenetic modifications, which can directly influence oligodendroglial biology.

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Jurkiewicz, Dorota, Elżbieta Ciara, Małgorzata Krajewska-Walasek, and Krystyna Chrzanowska. "DNA methylation as an epigenetic biomarker in imprinting disorders." Postępy Higieny i Medycyny Doświadczalnej 74 (December 7, 2020): 532–40. http://dx.doi.org/10.5604/01.3001.0014.5687.

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Epigenetic modifications control gene expression and enable the same genotype to lead to various phenotypes, thus exhibiting extensive variability in human cells function. DNA methylation is one of the most often investigated epigenetic modifications, playing a key part in genomic imprinting. Genomic imprinting is an epigenetic process by which the male and the female germ cells confer specific marks (imprints). Maternal chromatin marks differ from paternal ones, leading to expression of specific genes from only one allele. Disturbance in imprinting process results in epimutations, which are epigenetic defects, including DNA methylation changes. These abnormalities are identified in a group of imprinting disorders, associated with abnormal growth, development, behaviour and metabolism. Epimutations can occur spontaneously without any accompanying variant in DNA genomic sequence (a primary epimutation), whose defect can be a result of environmental factors. They can also be caused by changes in DNA sequence of genes involved in imprinting process (a secondary epimutation). DNA methylation in imprinting control regions is a very useful epigenetic biomarker and its detection is applied in the diagnostics of imprinting disorders. At present, various techniques for DNA methylation analysis are employed, which allow for investigations of one to several imprinted loci or the whole genome. DNA methylation studies are important not only in medical molecular diagnostics but are crucial in the search for therapies that would restore normal epigenetic status in patients.

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Zhang, Zhichao, Adeel Manaf, Yanjiao Li, Sonia Peña Perez, Rajikala Suganthan, John Arne Dahl, Magnar Bjørås, and Arne Klungland. "Histone Methylations Define Neural Stem/Progenitor Cell Subtypes in the Mouse Subventricular Zone." Molecular Neurobiology 57, no. 2 (October 25, 2019): 997–1008. http://dx.doi.org/10.1007/s12035-019-01777-5.

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Abstract Neural stem/progenitor cells (NSPCs) persist in the mammalian brain throughout life and can be activated in response to the physiological and pathophysiological stimuli. Epigenetic reprogramming of NPSC represents a novel strategy for enhancing the intrinsic potential of the brain to regenerate after brain injury. Therefore, defining the epigenetic features of NSPCs is important for developing epigenetic therapies for targeted reprogramming of NSPCs to rescue neurologic function after injury. In this study, we aimed at defining different subtypes of NSPCs by individual histone methylations. We found the three histone marks, histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 trimethylation (H3K27me3), and histone H3 lysine 36 trimethylation (H3K36me3), to nicely and dynamically portray individual cell types during neurodevelopment. First, we found all three marks co-stained with NSPC marker SOX2 in mouse subventricular zone. Then, CD133, Id1, Mash1, and DCX immunostaining were used to define NSPC subtypes. Type E/B, B/C, and C/A cells showed high levels of H3K27me3, H3K36me3, and H3K4me3, respectively. Our results reveal defined histone methylations of NSPC subtypes supporting that epigenetic regulation is critical for neurogenesis and for maintaining NSPCs.

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Zaidi, Sayyed K., Daniel W. Young, Martin Montecino, Jane B. Lian, Janet L. Stein, Andre J. van Wijnen, and Gary S. Stein. "Architectural Epigenetics: Mitotic Retention of Mammalian Transcriptional Regulatory Information." Molecular and Cellular Biology 30, no. 20 (August 9, 2010): 4758–66. http://dx.doi.org/10.1128/mcb.00646-10.

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ABSTRACT Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control the cellular phenotype by regulating gene expression. Recent results suggest that the mitotic retention of nuclease hypersensitivity, selective histone marks, as well as the lineage-specific transcription factor occupancy of promoter elements contribute to the epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain the control of phenotype and proliferation in progeny cells by bookmarking genes for activation or suppression.

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Motti, Maria Letizia, and Rosaria Meccariello. "Minireview: The Epigenetic Modulation of KISS1 in Reproduction and Cancer." International Journal of Environmental Research and Public Health 16, no. 14 (July 22, 2019): 2607. http://dx.doi.org/10.3390/ijerph16142607.

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Epigenetics describes how both lifestyle and environment may affect human health through the modulation of genome functions and without any change to the DNA nucleotide sequence. The discovery of several epigenetic mechanisms and the possibility to deliver epigenetic marks in cells, gametes, and biological fluids has opened up new perspectives in the prevention, diagnosis, and treatment of human diseases. In this respect, the depth of knowledge of epigenetic mechanisms is fundamental to preserving health status and to developing targeted interventions. In this minireview, we summarize the epigenetic modulation of the KISS1 gene in order to provide an example of epigenetic regulation in health and disease.

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Ozyerli-Goknar, Ezgi, and Tugba Bagci-Onder. "Epigenetic Deregulation of Apoptosis in Cancers." Cancers 13, no. 13 (June 27, 2021): 3210. http://dx.doi.org/10.3390/cancers13133210.

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Cancer cells possess the ability to evade apoptosis. Genetic alterations through mutations in key genes of the apoptotic signaling pathway represent a major adaptive mechanism of apoptosis evasion. In parallel, epigenetic changes via aberrant modifications of DNA and histones to regulate the expression of pro- and antiapoptotic signal mediators represent a major complementary mechanism in apoptosis regulation and therapy response. Most epigenetic changes are governed by the activity of chromatin modifying enzymes that add, remove, or recognize different marks on histones and DNA. Here, we discuss how apoptosis signaling components are deregulated at epigenetic levels, particularly focusing on the roles of chromatin-modifying enzymes in this process. We also review the advances in cancer therapies with epigenetic drugs such as DNMT, HMT, HDAC, and BET inhibitors, as well as their effects on apoptosis modulation in cancer cells. Rewiring the epigenome by drug interventions can provide therapeutic advantage for various cancers by reverting therapy resistance and leading cancer cells to undergo apoptotic cell death.

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Kent, Clement, and Pavan Agrawal. "Regulation of Social Stress and Neural Degeneration by Activity-Regulated Genes and Epigenetic Mechanisms in Dopaminergic Neurons." Molecular Neurobiology 57, no. 11 (August 3, 2020): 4500–4510. http://dx.doi.org/10.1007/s12035-020-02037-7.

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Abstract Transcriptional and epigenetic regulation of both dopaminergic neurons and their accompanying glial cells is of great interest in the search for therapies for neurodegenerative disorders such as Parkinson’s disease (PD). In this review, we collate transcriptional and epigenetic changes identified in adult Drosophila melanogaster dopaminergic neurons in response to either prolonged social deprivation or social enrichment, and compare them with changes identified in mammalian dopaminergic neurons during normal development, stress, injury, and neurodegeneration. Surprisingly, a small set of activity-regulated genes (ARG) encoding transcription factors, and a specific pattern of epigenetic marks on gene promoters, are conserved in dopaminergic neurons over the long evolutionary period between mammals and insects. In addition to their classical function as immediate early genes to mark acute neuronal activity, these ARG transcription factors are repurposed in both insects and mammals to respond to chronic perturbations such as social enrichment, social stress, nerve injury, and neurodegeneration. We suggest that these ARG transcription factors and epigenetic marks may represent important targets for future therapeutic intervention strategies in various neurodegenerative disorders including PD.

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Joglekar, Mugdha V., Vinay M. Joglekar, Sheela V. Joglekar, and Anandwardhan A. Hardikar. "Human fetal pancreatic insulin-producing cells proliferate in vitro." Journal of Endocrinology 201, no. 1 (January 26, 2009): 27–36. http://dx.doi.org/10.1677/joe-08-0497.

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There have been considerable efforts towards understanding the potential of human pancreatic endocrine cells to proliferate and transition into mesenchymal cell populations. Since rodent studies have demonstrated that mouse insulin-producing cells do not proliferate in vitro, a similar possibility has been considered for human islet endocrine cells. Considering the inherent differences in mouse and human pancreatic islets, we decided to assess the potential of human fetal pancreatic insulin-producing cells to proliferate in vitro. We studied the proliferative potential of human fetal pancreatic islet-derived populations from second or third trimester fetal pancreas and characterized the cells that grow out during their expansion. We have used seven different approaches including in situ hybridization and immunostaining, quantitative estimation of multiple gene transcripts in populations as well as in single cells, clonal analysis of islet cells, assessment of heritable marks of active insulin promoter, and thymidine analog-based lineage tracing. Our studies demonstrate that human fetal pancreatic insulin-producing cells proliferate in vitro to generate mesenchymal cell populations. Interestingly, epigenetic modifications that mark open chromatin conformation of insulin promoter regions are retained even after a million fold expansion/proliferation in vitro. These findings demonstrate that hormone-producing cells in pancreatic islets proliferate in vitro and retain epigenetic marks that characterize an active insulin promoter. Such in vitro-derived mesenchymal cells may be of potential use in cell-replacement therapy for diabetes.

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Inoue, Daichi, Takeshi Fujino, and Toshio Kitamura. "ASXL1 as a critical regulator of epigenetic marks and therapeutic potential of mutated cells." Oncotarget 9, no. 81 (October 16, 2018): 35203–4. http://dx.doi.org/10.18632/oncotarget.26230.

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Shiota, Kunio, Yasushi Kogo, Jun Ohgane, Takuya Imamura, Atsushi Urano, Koichiro Nishino, Satoshi Tanaka, and Naka Hattori. "Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice." Genes to Cells 7, no. 9 (September 2002): 961–69. http://dx.doi.org/10.1046/j.1365-2443.2002.00574.x.

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Journal articles on the topic 'MEL cells; Epigenetic marks'

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