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Polioudaki, Hara, Yolanda Markaki, Niki Kourmouli, George Dialynas, Panayiotis A. Theodoropoulos, Prim B. Singh, and Spyros D. Georgatos. "Mitotic phosphorylation of histone H3 at threonine 3." FEBS Letters 560, no. 1-3 (February 4, 2004): 39–44. http://dx.doi.org/10.1016/s0014-5793(04)00060-2.
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Hurd, Paul J., Andrew J. Bannister, Karen Halls, Mark A. Dawson, Michiel Vermeulen, Jesper V. Olsen, Heba Ismail, et al. "Phosphorylation of Histone H3 Thr-45 Is Linked to Apoptosis." Journal of Biological Chemistry 284, no. 24 (April 10, 2009): 16575–83. http://dx.doi.org/10.1074/jbc.m109.005421.
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Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-δ as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.
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Wang, Zhen, Juan Armando Casas-Mollano, Jianping Xu, Jean-Jack M. Riethoven, Chi Zhang, and Heriberto Cerutti. "Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana." Proceedings of the National Academy of Sciences 112, no. 27 (June 22, 2015): 8487–92. http://dx.doi.org/10.1073/pnas.1423325112.
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Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5′ genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).
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Żabka, Aneta, Natalia Gocek, Konrad Winnicki, Paweł Szczeblewski, Tomasz Laskowski, and Justyna Teresa Polit. "Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions." Cells 10, no. 12 (December 3, 2021): 3409. http://dx.doi.org/10.3390/cells10123409.
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Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.
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Yoshida, Makoto M., Lily Ting, Steven P. Gygi, and Yoshiaki Azuma. "SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis." Journal of Cell Biology 213, no. 6 (June 20, 2016): 665–78. http://dx.doi.org/10.1083/jcb.201511079.
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DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.
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Karakkat, Jimsheena V., Suneesh Kaimala, Sreejisha P. Sreedharan, Princy Jayaprakash, Ernest A. Adeghate, Suraiya A. Ansari, Ernesto Guccione, Eric P. K. Mensah-Brown, and Bright Starling Emerald. "The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex." Nucleic Acids Research 47, no. 19 (September 16, 2019): 10086–103. http://dx.doi.org/10.1093/nar/gkz786.
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Abstract The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.
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Kang, Hyoeun, Yong Seok Park, Dong-Hyung Cho, Jae-Sung Kim, and Jeong Su Oh. "Dynamics of histone H3 phosphorylation at threonine 3 during meiotic maturation in mouse oocytes." Biochemical and Biophysical Research Communications 458, no. 2 (March 2015): 280–86. http://dx.doi.org/10.1016/j.bbrc.2015.01.099.
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Wang, Fangwei, Natalia P. Ulyanova, John R. Daum, Debasis Patnaik, Anna V. Kateneva, Gary J. Gorbsky, and Jonathan M. G. Higgins. "Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation." Journal of Cell Biology 199, no. 2 (October 15, 2012): 251–68. http://dx.doi.org/10.1083/jcb.201205106.
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Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.
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Quadri, Roberto, Sarah Sertic, Anna Ghilardi, Diego Rondelli, Guido Roberto Gallo, Luca Del Giacco, and Marco Muzi-Falconi. "Phosphorylation of H3-Thr3 by Haspin Is Required for Primary Cilia Regulation." International Journal of Molecular Sciences 22, no. 14 (July 20, 2021): 7753. http://dx.doi.org/10.3390/ijms22147753.
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Primary cilia are commonly found on most quiescent, terminally differentiated cells and play a major role in the regulation of the cell cycle, cell motility, sensing, and cell–cell communication. Alterations in ciliogenesis and cilia maintenance are causative of several human diseases, collectively known as ciliopathies. A key determinant of primary cilia is the histone deacetylase HDAC6, which regulates their length and resorption and whose distribution is regulated by the death inducer-obliterator 3 (Dido3). Here, we report that the atypical protein kinase Haspin is a key regulator of cilia dynamics. Cells defective in Haspin activity exhibit longer primary cilia and a strong delay in cilia resorption upon cell cycle reentry. We show that Haspin is active in quiescent cells, where it phosphorylates threonine 3 of histone H3, a known mitotic Haspin substrate. Forcing Dido3 detachment from the chromatin prevents Haspin inhibition from impacting cilia dynamics, suggesting that Haspin activity is required for the relocalization of Dido3–HDAC6 to the basal body. Exploiting the zebrafish model, we confirmed the physiological relevance of this mechanism. Our observations shed light on a novel player, Haspin, in the mechanisms that govern the determination of cilia length and the homeostasis of mature cilia.
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Nguyen, A. L., A. S. Gentilello, A. Z. Balboula, V. Shrivastava, J. Ohring, and K. Schindler. "Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes." Journal of Cell Science 127, no. 23 (October 14, 2014): 5066–78. http://dx.doi.org/10.1242/jcs.158840.
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Cao, Zubing, Tengteng Xu, Xu Tong, Dandan Zhang, Chengxue Liu, Yiqing Wang, Di Gao, et al. "HASPIN kinase mediates histone deacetylation to regulate oocyte meiotic maturation in pigs." Reproduction 157, no. 6 (June 2019): 501–10. http://dx.doi.org/10.1530/rep-18-0447.
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HASPIN kinase-catalyzed phosphorylation of histone H3 on threonine 3 (H3T3p) directs the activity and localization of chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC) to regulate chromosome condensation and segregation in both mitosis and meiosis. However, the function of HASPIN kinase in the meiotic maturation of porcine oocytes is not yet known. Here, we found that HASPIN mRNA is constantly expressed in porcine oocyte maturation and subsequent early embryo development. H3T3p is highly enriched on chromosomes at germinal vesicle breakdown (GVBD) stage and thereafter maintains a low level in progression through metaphase I (MI) to metaphase II (MII). Correspondingly, H3T3p was completely abolished in oocytes treated with an inhibitor of HASPIN kinase. Functionally, inhibition of HASPIN activity led to a significant reduction in the rate of oocyte meiotic maturation and the limited cumulus expansion. Additionally, HASPIN inhibition caused both spindle disorganization and chromosome misalignment in oocytes at MI and MII stage. Importantly, HASPIN inhibition severely prevented deacetylation of several highly conserved lysine (K) residues of histone H3 and H4 including H3K9, H3K14, H4K5, H4K8, H4K12 and H4K16 on the metaphase chromosomes during oocyte meiotic maturation. Taken together, these results demonstrate that HASPIN kinase regulates porcine oocyte meiotic maturation via modulating histone deacetylation.
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Edgerton, Heather, Marnie Johansson, Daniel Keifenheim, Soumya Mukherjee, Jeremy M. Chacón, Jeff Bachant, Melissa K. Gardner, and Duncan J. Clarke. "A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis." Journal of Cell Biology 213, no. 6 (June 20, 2016): 651–64. http://dx.doi.org/10.1083/jcb.201511080.
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Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin–histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.
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Karimi-Ashtiyani, Raheleh, and Andreas Houben. "In Vitro Phosphorylation of Histone H3 at Threonine 3 by Arabidopsis Haspin Is Strongly Influenced by Posttranslational Modifications of Adjacent Amino Acids." Molecular Plant 6, no. 2 (March 2013): 574–76. http://dx.doi.org/10.1093/mp/sss149.
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Fujimura, Akiko, Yuki Hayashi, Kazashi Kato, Yuichiro Kogure, Mutsuro Kameyama, Haruka Shimamoto, Hiroaki Daitoku, Akiyoshi Fukamizu, Toru Hirota, and Keiji Kimura. "Identification of a novel nucleolar protein complex required for mitotic chromosome segregation through centromeric accumulation of Aurora B." Nucleic Acids Research 48, no. 12 (June 1, 2020): 6583–96. http://dx.doi.org/10.1093/nar/gkaa449.
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Abstract The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.
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Thakar, Sumukh, Yash T. Katakia, Shyam Kumar Ramakrishnan, Niyati Pandya Thakkar, and Syamantak Majumder. "Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation." Cells 10, no. 10 (September 26, 2021): 2548. http://dx.doi.org/10.3390/cells10102548.
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Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions—namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2′s recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.
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Nishida-Fukuda, Hisayo, Keizo Tokuhiro, Yukio Ando, Hiroaki Matsushita, Morimasa Wada, and Hiromitsu Tanaka. "Evaluation of the antiproliferative effects of the HASPIN inhibitor CHR-6494 in breast cancer cell lines." PLOS ONE 16, no. 4 (April 14, 2021): e0249912. http://dx.doi.org/10.1371/journal.pone.0249912.
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HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.
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Hoek, Maarten, Michael P. Myers, and Bruce Stillman. "An Analysis of CAF-1-interacting Proteins Reveals Dynamic and Direct Interactions with the KU Complex and 14-3-3 Proteins." Journal of Biological Chemistry 286, no. 12 (January 5, 2011): 10876–87. http://dx.doi.org/10.1074/jbc.m110.217075.
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CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.
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Robert, F., C. Verschraegen, H. Hurwitz, H. Uronis, R. Advani, A. Chen, P. Taverna, M. Wollman, J. Fox, and G. Michelson. "A phase I trial of sns-314, a novel and selective pan-aurora kinase inhibitor, in advanced solid tumor patients." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 2536. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.2536.
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2536 Background: Aurora Kinases are a family of serine/threonine kinases (Aurora Kinases (AK) A, B, and C) critical for mitosis. Elevated AKs expression occurs in a high percentage of melanoma, colon, breast, ovarian, gastric, and pancreatic tumors; in a subset of these tumors the AURKA locus (20q13) is amplified. SNS-314 is a selective pan-AK inhibitor with low nanomolar IC50s. Methods: Study design is 3+3 phase 1 dose escalation by modified Fibonacci. Patients (pts) with advanced solid tumors received SNS-314 by 3 hour infusion qweek X 3 (28 day cycle). Primary endpoints: safety, tolerability, and DLT assessment. Secondary endpoints: MTD, pharmacokinetics (PK), pharmacodynamics, and antitumor activity. Pharmacodynamic endpoint was inhibition of Histone H3 phosphorylation (pHH3) evaluated by immunohistochemistry of skin punch biopsies taken pre- and 2 hours post-infusion. Results: Thirty-two pts (16M/16F; median age = 58.5 years) were enrolled into 8 cohorts: dose range 30–1800 mg/m2. Median cycles received =2. SNS-314 was generally well tolerated with Grade 1–2 toxicities ≥ 15% incidence: nausea (31%), fatigue (28%), vomiting, constipation, and pain (16% each), and no Grade 3+ toxicities of ≥ 15% incidence. A DLT of Grade 3 neutropenia preventing administration of all 3 doses was observed at 1440 mg/m2. Plasma PK were dose proportional for exposure with no accumulation of SNS-314 following weekly administration. Clearance was moderate (5.65 L/hr/m2, CV 39.4%); Vss approximated total body water (21.5 L/m2, CV 78.1%); terminal half-life was 10.4 hours (CV 66.8%). Six patients had stable disease as their best response. Inhibition of pHH3 by SNS-314 was observed in skin biopsies of patients treated at doses of 240 mg/m2 and greater. Conclusions: SNS-314 is a novel inhibitor of AKs A, B, and C. The compound has been generally well tolerated; MTD was not established. No objective responses were observed. Pharmacodynamic activity was demonstrated by inhibition of pHH3. [Table: see text]
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Kristeleit, R., H. Calvert, H. Arkenau, D. Olmos, J. Adam, E. R. Plummer, V. Lock, M. Squires, L. Fazal, and I. Judson. "A phase I study of AT9283, an aurora kinase inhibitor, in patients with refractory solid tumors." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 2566. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.2566.
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2566 Background: AT9283, a multitargeted kinase inhibitor, inhibits several closely related tyrosine and serine/threonine kinases with an IC50 of <10 nM including Aurora A and B, JAK and ABL. Exposure of solid tumour cell lines to AT9283 in vitro induces an “aurora inhibitory” phenotype. Cell survival decreases with increased duration of exposure. Methods: A phase I dose escalation study was performed using a 72 hour continuous intravenous (iv) infusion schedule repeated three weekly according to a stand ard “3+3” design. Results: Thirty-three patients have been treated with a median age of 61 (range 33 to 76 years). The maximum tolerated dose (MTD) was 9 mg/m2/day. Treatment was well tolerated with febrile neutropenia the only dose limiting toxicity. Other adverse events considered possibly related to AT9283 were reversible and included gastrointestinal disturbance and fatigue. Biological evidence of aurora B inhibition manifest as a reduction in histone H3 phosphorylation in skin biopsies during the infusion was observed at all dose levels. A plateau steady state plasma concentration of AT9283 was achieved within 24 hours of initiating drug infusion at all dose levels and exposure increased linearly with dose. Seven patients received an initial oral dose of AT9283 as an aqueous solution in a fasting state at a dose of 0.9 mg mg/m2 one week prior to starting iv treatment. Interim pharmacokinetic analysis indicated that the median oral bioavailability was 27% (range 17 to 45%) The best response to treatment was a partial response in one patient with NSCLC (ongoing). An additional 4 patients received at least six cycles of therapy (squamous cell carcinoma of the lung, adenocarcinoma of the esophagus and colorectal carcinoma [2]) with a best response of stable disease. Conclusions: The MTD of AT9283 when administered as a 72 hour continuous iv infusion was 9mg/m2/day. Febrile neutropenia is the dose limiting toxicity and evidence of anticancer activity was seen in heavily pre-treated patients. [Table: see text] [Table: see text]
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Hammond, Sharra, Stephanie Byrum, Sarita Namjoshi, Hilary Graves, briana Dennehey, Alan J. Tackett, and Jessica Tyler. "Mitotic phosphorylation of histone H3 threonine 80." Cell Cycle 13, no. 3 (November 25, 2013): 440–52. http://dx.doi.org/10.4161/cc.27269.
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Okabe, Seiichi, Tetsuzo Tauchi, Yuko Tanaka, Toshihiko Kitahara, and Kazuma Ohyashiki. "Efficacy Of The Polo-Like Kinase Inhibitor, Rigosertib Alone Or In Combination With ABL Tyrosine Kinase Inhibitor Against BCR-ABL-Positive Leukemia Cells." Blood 122, no. 21 (November 15, 2013): 3985. http://dx.doi.org/10.1182/blood.v122.21.3985.3985.
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Abstract Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. Polo-like kinases (PLK) are the family of serine threonine kinases and essential for mitosis. PLK is also critical regulator of cell cycle progression and DNA damage response. One of the PLK and phosphoinositide 3-kinase (PI3K) inhibitor, rigosertib (ON 01910.Na) is a novel synthetic benzyl styryl sulfone that is cytotoxic against a variety of human tumor cell lines. Rigosertib is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies such as myelodysplastic syndromes (MDS). We suggested that rigosertib mediated inhibition PLK and PI3K activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. In this study, we investigated the combination therapy with a rigosertib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells. 72 h treatment of rigosertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of rigosertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We examined the intracellular signaling after treatment of rigosertib. Phosphorylation of BCR-ABL, Crk-L was not reduced. However, activity of caspase 3 was increased. We next investigated the efficacy between imatinib and rigosertib by using these cell line. Combined treatment of K562 cells with imatinib and rigosertib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP and γH2A.X phosphorylation was increased after imatinib and rigosertib treatment. Anti-apoptotic protein, Mcl-1 was also decreased. We also found the phosphorylation of histone H3 was increased after rigosertib treatment suggested that the cells arrested in G2/M phase. We investigated the rigosertib activity against T315I positive cells. Rigosertib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and rigosertib caused significantly more cytotoxicity than each drug alone.We next investigated by using ponatinib resistant Ba/F3 cells and nilotinib resistant K562 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of rigosertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells and K562 nilotinib resistant cells. These results indicated that administration of the PLK and PI3K inhibitor, rigosertib may be a powerful strategy against ABL TKI resistant cells and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.
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Fong, Jerry J., Brenda L. Nguyen, Robert Bridger, Estela E. Medrano, Lance Wells, Shujuan Pan, and Richard N. Sifers. "β-N-Acetylglucosamine (O-GlcNAc) Is a Novel Regulator of Mitosis-specific Phosphorylations on Histone H3." Journal of Biological Chemistry 287, no. 15 (February 27, 2012): 12195–203. http://dx.doi.org/10.1074/jbc.m111.315804.
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O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.
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Mahadevan, Daruka, Wenqing Qi, Laurence Cooke, Xiabing Lui, Daniel Oscar Persky, Lisa M. Rimsza, and Thomas P. Miller. "Targeting Aurora Kinase in Aggressive B-Cell Non-Hodgkin's Lymphomas." Blood 114, no. 22 (November 20, 2009): 284. http://dx.doi.org/10.1182/blood.v114.22.284.284.
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Abstract Abstract 284 Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Aurora kinases during the cell cycle over-rides mitotic and spindle check points leading to aneuploidy in many human cancers; Aurora kinases are therefore attractive therapeutic targets. Gene expression profiling in aggressive B- and T-cell non-Hodgkin's lymphoma (NHL) has shown the Aurora kinases to be over-expressed and they may be key component genes of the ‘proliferative' signature. We hypothesized (1) Aurora kinases are over-expressed in human aggressive B-cell NHL (mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (TFL)), (2) Aurora ATP-binding site small molecule inhibitor (SMI) is effective in promoting apoptosis in cell culture and tumor growth inhibition (TGI) in mouse xenograft model(s) of NHL, and (3) Aurora SMI will be safe and effective in treating patients with relapsed aggressive B-cell NHL in early phase clinical trials. To analyze Aurora expression, tissue microarrays (TMA) were constructed from 43 patients with DLBCL and 40 with MCL, and Aurora A and B expression optimized with commercially available antibodies by immunohistochemistry (IHC). The IHC was rated as a staining intensity on a scale of 0 to 3+. The NHL TMAs demonstrated intense staining (2+ to 3+) for Aurora A (nucleus) and Aurora B (nucleus) in >60% compared to normal lymph nodes. We also analyzed the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) publicly available database for Aurora A and B expression in MCL and found a worse survival in those with A > B over-expression (p<0.01). Since both Auroras are transforming genes, the LLMPP data support the conclusion that these S/T kinases are associated with a poor prognosis and are potential targets for therapy. Western blotting analysis of 13 B-cell NHL cell lines (DLBCL, MCL and TFL) for Aurora A and B expression showed significant over-expression compared to B-cells isolated from normal lymph nodes. Aurora A knockdown by shRNA in the B-NHL cell lines showed inhibition of mitosis with a polyploid phenotype (4n, 8n) that ends in apoptosis as shown by PARP-cleavage. The Aurora A specific inhibitor (MLN8237) evaluated in the 13 NHL cell lines phenocopies shRNA knockdown with associated inhibition of proliferation (IC50=0.05 mM) and promotes apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Combination of MLN8237 with a microtubule targeting agent (docetaxel) to abrogate the spindle checkpoint is synergistic in a sequence specific manner. Moreover, MLN8237 effectively inhibits Aurora A auto-phosphorylation and eliminates phospho-histone H3 (Ser10) phosphorylation. Currently, 2 mouse MCL (Granta 519) xenograft models are underway evaluating tumor growth inhibition (TGI), safety and survival of MLN8237 alone and in combination with docetaxel or rituximab respectively. Preliminary data show that MLN8237 is synergistic with docetaxel in TGI in the MCL xenograft model. Together the data suggest inhibition of Aurora kinases may offer a promising treatment strategy for patients with aggressive B-cell NHL [Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.]. Disclosures: Rimsza: High Throughput Genomics:.
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Martinez, Danielle R., Hunter W. Richards, Qiushi Lin, Carlos A. Torres-Cabala, Victor G. Prieto, Jonathan L. Curry, and Estela E. Medrano. "H3K79me3T80ph is a Novel Histone Dual Modification and a Mitotic Indicator in Melanoma." Journal of Skin Cancer 2012 (2012): 1–9. http://dx.doi.org/10.1155/2012/823534.
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The current study characterizes the mitosis-associated histone dual modification on the core of histone H3: trimethylation of histone H3 lysine 79 and simultaneous phosphorylation of H3 threonine 80 (H3K79me3T80ph). Through the use of protein and microscopy-based techniques, we find that H3K79me3T80ph shares a similar spatial and temporal regulation as H3S10ph but additionally requires methyltransferase activity. In addition, we find that Aurora kinase activity is necessary for the catalysis of H3K79me3T80phin vivo. Finally, our analysis of H3K79me3T80ph using a tissue microarray indicates that H3K79me3T80ph marks a subset of primary cutaneous melanomas with metastatic potential indicating that H3K79me3T80ph may identify a subset of invasive melanomas with a more aggressive clinical behaviour.
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Xie, Jing, Matthew Wooten, Vuong Tran, Bi-Chang Chen, Caitlin Pozmanter, Christine Simbolon, Eric Betzig, and Xin Chen. "Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline." Cell 163, no. 4 (November 2015): 920–33. http://dx.doi.org/10.1016/j.cell.2015.10.002.
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Zeitlin, S. G., C. M. Barber, C. D. Allis, and K. Sullivan. "Differential regulation of CENP-A and histone H3 phosphorylation in G2/M." Journal of Cell Science 114, no. 4 (February 15, 2001): 653–61. http://dx.doi.org/10.1242/jcs.114.4.653.
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After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.
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Chen, Lisa S., William G. Wierda, Sanjeev Redkar, David J. Bearss, and Varsha Gandhi. "Pim Kinase Inhibitor, SGI-1776, Induces Apoptosis in CLL Lymphocytes." Blood 112, no. 11 (November 16, 2008): 4199. http://dx.doi.org/10.1182/blood.v112.11.4199.4199.
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Abstract We report investigational studies of a Pim-1 kinase inhibitor, SGI-1776, as a therapeutic agent for the treatment of chronic lymphocytic leukemia (CLL). Pim family proteins are serine/threonine kinase inhibitors of apoptosis, and elevated expression of Pim kinases have been detected in hematological malignancies and in certain solid tumors. Three Pim kinases have been identified to date, Pim-1, -2 and -3. Pim-1 has been shown to synergize with c-Myc in tumorigenesis, and increased expression of the human Pim-2 gene is observed in CLL and non-Hodgkin lymphomas. Specifically, Pim-1 and Pim-2 have been shown to be required for efficient pre–B-cell transformation by v-Abl oncogene. Small molecule SGI-1776 was evaluated using Ambit Biosciences’ KINOMEscan and was found to have IC50s in the nanomolar range for Pim-1, Pim-2, and Pim-3 using the Millipore IC50 Profiler. SGI-1776 was screened against a panel of kinases utilizing radiolabeled biochemical assays, and was found to be highly selective for Pim kinases without any effects on CDKs, Chk1, IKK, JNK, Abl, Raf, MAP kinases and protein kinase A and B. Specifically, the IC50 were measured as follows: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM.) The other two enzymes affected at nM concentration of SGI-1776 were Flt-2 and haspin. Using primary samples obtained from patients with CLL, we evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of primary CLL cells (n=7), with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 10%, 22% and 38% respectively, compared with untreated cells. SGI-1776-induced apoptosis was observed in heterogeneous patient populations, and there was disparity in the expression levels of traditional CLL prognostic markers including ZAP-70, b2-microglobulin, IgVH mutation status, Rai stage and number of prior treatments. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10) were not affected by SGI-1776 treatment in CLL, unlike in replicating cell types, suggesting an alternative mechanism in CLL. We then evaluated the potential inhibition of c-Myc driven transcription by measuring total RNA synthesis. Following treatment with 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to approximately 50% of control, measured using a radioactive uridine incorporation assay (n = 3). There was also a reduction in Mcl-1 and c-Myc protein level, both of which have short transcript half-lives. The reduction in Mcl-1 protein was not due to apoptosis or cleavage by caspases, since Mcl-1 reduction occurred in the presence of caspase inhibitor ZVAD, nor was there an increase in cleaved Mcl-1. In contrast, there was no change in anti-apoptotic proteins Bcl-2, XIAP, survivin, stabilization of p53 or p21. Taken together, SGI-1776 consistently induced apoptosis in CLL cells. Although its mechanism of action is not fully elucidated, inhibition of RNA synthesis and reduction of Mcl-1 and c-Myc protein levels are associated with SGI-1776-induced apoptosis.
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Sun, Guangyan C., Anna C. Shvab, Bin C. Li, Felipe C. Beckedorff, Guy Jacques Leclerc, Ramin Shiekhattar, and Julio C. Barredo. "Acadesine Elicits a Rapid Epigenetic Reprograming of Immediate Early Genes through the Protein Kinase D1 Pathway in Acute Lymphoblastic Leukemia Cells Undergoing Energy/Metabolic Stress." Blood 132, Supplement 1 (November 29, 2018): 1321. http://dx.doi.org/10.1182/blood-2018-99-112678.
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Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance. Disclosures No relevant conflicts of interest to declare.
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Bui, H. T., V. T. Nguyen, T. Wakayama, and T. Miyano. "123 HISTONE H3 MODIFICATIONS IN PIG OOCYTES DURING GROWTH, MATURATION, AND ACTIVATION." Reproduction, Fertility and Development 18, no. 2 (2006): 170. http://dx.doi.org/10.1071/rdv18n2ab123.
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Oocyte growth, maturation, and activation are complex processes that include transcription, heterochromatin formation, chromosome condensation and decondensation, two consecutive chromosome separations, and genomic imprinting for producing the mature egg. The first sign of oocyte maturation is phosphorylation of histone H3, which leads to the chromosome condensation (Bui et al. 2004 Biol. Reprod. 70, 1843-1851). The objective of this study was to investigate the change in chromosome morphology in relation to histone modifications in pig oocytes during growth, maturation, and activation. Growing oocytes were collected from follicles at various diameters (from 0.1 to 6 mm) in pig ovaries. For maturation, oocyte-cumulus-granulosa cell complexes (OCGC) were collected from follicles that were 3 to 6 mm in diameter and cultured in modified TCM 199 for different periods of time to obtain meiotic stages of oocytes. For activation, oocytes were cultured for maturation in 42 h and were activated using a protocol that was described previously (Nguyen et al. 2003 Theriogenology 59, 719-734). Then, oocytes were examined by immunostaining with antibodies: anti-phospho-histone H3 at serine 10 or serine 28 (S10 or S28), anti-trimethyl-histone H3 at lysine 9 (K9), and anti-acetyl-histone H3 at lysine 9, 14, or 28 (K9, K14, or K28). Some oocytes were examined for double assay of Cdc2 and H3 kinase, which were measured by phosphorylation of histone H1 and myelin basic protein as their substrates. To examine the effects of histone deacetylase (HDAC) inhibition, OCGC were cultured in maturation medium supplemented with or without 100 nM trichostatin A for 42 h. The results show that, during the growth phase, histone H3 became methylated at K9 and is acetylated at K9, K14, and K18. When the fully grown oocytes start maturation, histone H3 becomes phosphorylated at S28 and then S10 and is deacetylated at K9, K14, and K18. After oocyte activation, reacetylation and dephosphorylation of histone H3 correlates to the decondensation of chromosomes. We also found that the activity of histone H3 kinase occurred at a similar time course to that of phosphorylation of histone H3-S28. This suggests that phosphorylation of H3-S28 might be one of the key events initiating meiotic chromosome condensation. The inhibition of HDAC induces maintenance of acetylation of H3-K14 and dephosphorylation of histone H3 at S10 and S28. Therefore, the chromosome could not condense and affect meiotic progression. It is possible that deacetylation is required for the phosphorylation of histone H3. The results suggest that chromatin morphology of pig oocytes is regulated by acetylation/deacetylation and phosphorylation/dephosphorylation of histone H3 and that histone deacetylase activity is essential for the process of chromatin remodeling in pre-ovulatory oocytes. Although histone acetylation and phosphorylation were reversible, histone methylation has energetic stability and is established during the oocyte growth phase. It is also suggested that the ordered phosphorylation of histone H3 at S10 and S28 is influenced by acetylation of neighboring lysines in the histone H3 molecule.
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Waterborg, Jakob H. "Evolution of histone H3: emergence of variants and conservation of post-translational modification sites1This article is part of Special Issue entitled Asilomar Chromatin and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 90, no. 1 (February 2012): 79–95. http://dx.doi.org/10.1139/o11-036.
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Histone H3 proteins are highly conserved across all eukaryotes and are dynamically modified by many post-translational modifications (PTMs). Here we describe a method that defines the evolution of the family of histone H3 proteins, including the emergence of functionally distinct variants. It combines information from histone H3 protein sequences in eukaryotic species with the evolution of these species as described by the tree of life (TOL) project. This so-called TOL analysis identified the time when the few observed protein sequence changes occurred and when distinct, co-existing H3 protein variants arose. Four distinct ancient duplication events were identified where replication-coupled (RC) H3 variants diverged from replication-independent (RI) forms, like histone H3.3 in animals. These independent events occurred in ancestral lineages leading to the clades of metazoa, viridiplantae, basidiomycota, and alveolata. The proto-H3 sequence in the last eukaryotic common ancestor (LECA) was expanded to at least 133 of its 135 residues. Extreme conservation of known acetylation and methylation sites of lysines and arginines predicts that these PTMs will exist across the eukaryotic crown phyla and in protists with canonical chromatin structures. Less complete conservation was found for most serine and threonine phosphorylation sites. This study demonstrates that TOL analysis can determine the evolution of slowly evolving proteins in sequence-saturated datasets.
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Walter, Wendy, David Clynes, Yong Tang, Ronen Marmorstein, Jane Mellor, and Shelley L. Berger. "14-3-3 Interaction with Histone H3 Involves a Dual Modification Pattern of Phosphoacetylation." Molecular and Cellular Biology 28, no. 8 (February 11, 2008): 2840–49. http://dx.doi.org/10.1128/mcb.01457-07.
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ABSTRACT Histone modifications occur in precise patterns and are proposed to signal the recruitment of effector molecules that profoundly impact chromatin structure, gene regulation, and cell cycle events. The linked modifications serine 10 phosphorylation and lysine 14 acetylation on histone H3 (H3S10phK14ac), modifications conserved from Saccharomyces cerevisiae to humans, are crucial for transcriptional activation of many genes. However, the mechanism of H3S10phK14ac involvement in these processes is unclear. To shed light on the role of this dual modification, we utilized H3 peptide affinity assays to identify H3S10phK14ac-interacting proteins. We found that the interaction of the known phospho-binding 14-3-3 proteins with H3 is dependent on the presence of both of these marks, not just phosphorylation alone. This is true of mammalian 14-3-3 proteins as well as the yeast homologues Bmh1 and Bmh2. The importance of acetylation in this interaction is also seen in vivo, where K14 acetylation is required for optimal Bmh1 recruitment to the GAL1 promoter during transcriptional activation.
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Bui Hong, T., L. G. Villa-Diaz, E. Yamaoka, and T. Miyano. "309CHROMOSOME CONDENSATION IS CORRELATED WITH HISTONE H3 PHOSPHORYLATION WITHOUT CDC2 KINASE AND MAP KINASE ACTIVITIES IN PIG OOCYTES." Reproduction, Fertility and Development 16, no. 2 (2004): 273. http://dx.doi.org/10.1071/rdv16n1ab309.
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Chromosome condensation is the first step of oocyte maturation. When the oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine (1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during meiotic maturation of pig oocytes, and (2) the effects of protein phosphatase 1/2A (PP1/PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase and histone H3 kinase in this process. Oocyte-cumulus-granulosa cell complexes (OCGCs) were collected from follicles of 4–6mm in diameter. OCGCs were cultured in modified TCM 199 for different periods of time to obtain oocytes at the germinal vesicle (GV, 0h), diakinesis (18h), metaphase I (24–27h), anaphase I to telophase I (30–33h), and metaphase II (42h) stages. To examine the effects of PP1/PP2A inhibitors on the chromosome condensation, oocyte-cumulus-complexes (OCCs) were cultured in modified TCM 199 with either 2.5μM okadaic acid (OA) or 50nM calyculin A (CL-A) for 0.5, 1, 2, 3, 4 and 6h. To inhibit the MAP kinase activity in the oocytes treated with the PP1/PP2A inhibitor, OCCs were cultured in medium containing CL-A and the MEK inhibitor, U0126 (0.1mM). Morphology of the chromosome and nuclear membrane, and phosphorylation of histone H3 were examined by the immunofluorescent microscopy. In each group 30 oocytes were examined for OA or CL-A and 60 oocytes for CL-A+U0126 treatments. Activities of Cdc2 kinase, MAP kinase and histone H3 kinase were also examined. Phosphorylation of histone H3 (Ser10) was not detected in the oocytes at the GV stage. The phosphorylation was first detected in the clump of condensed chromosomes at the diakinesis stage of prophase I and maintained until metaphase II. The kinase assay also showed that histone H3 kinase activity was low in GV oocytes, increased at the diakinesis stage, and then maintained high activity until metaphase II. PP1/PP2A inhibitors induced rapid chromosome condensation in pig oocytes. Histone H3 phosphorylation (Ser10) became detectable together with the chromosome condensation in the treated oocytes after 2h. After 6h, oocytes had highly condensed chromosomes with phosphorylated histone H3 (81% in CL-A- and 71% in OA-treated oocytes). Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and U0126, neither Cdc2 kinase nor MAP kinase were activated, although histone H3 kinase was still activated and chromosomes condensed. These results suggest that phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Futhermore, the chromosome condensation is correlated with histone H3 kinase activity, but not with Cdc2 kinase and MAP kinase activities.
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Okabe, Seiichi, Tetsuzo Tauchi, Seiichiro Katagiri, Yuko Tanaka, and Kazuma Ohyashiki. "Activity of the Aurora Kinase Inhibitor, MLN8237 (alisertib) Alone or in Combination with Ponatinib Against Imatinib-Resistant BCR-ABL-Positive Cells." Blood 120, no. 21 (November 16, 2012): 1333. http://dx.doi.org/10.1182/blood.v120.21.1333.1333.
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Abstract Abstract 1333 Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor, imatinib has demonstrated the potency against CML patients. However, resistance to imatinib can develop in CML patients due to BCR-ABL point mutations. One of T315I mutation is resistant to currently available ABL tyrosine kinase inhibitors. Therefore, new approach against T315I mutant may improve the outcome of Ph-positive leukemia patients. Aurora kinases are serine/threonine kinases and upregulated in many malignancies including leukemia, and play an important role in cell cycle control and tumor proliferations. Because Aurora kinases are overexpressed in leukemia cells, Aurora kinases may present attractive targets for leukemia treatment. One of Aurora kinase inhibitor, MLN8237 (alisertib) is an oral and selective Aurora kinase A inhibitor and is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies. We suggested that alisertib mediated inhibition Aurora kinase activity and in combination with ponatinib, also known as AP24534 may abrogate the proliferation and survival of Ph-positive cells including T315I mutation. In this study, we investigated the combination therapy with a ponatinib and an alisertib by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, ponatinib resistant Ba/F3 cells and T315I primary sample. Protein expression of Aurora A and B were increased in Ph-positive leukemia cells. 72 hours treatment of alisertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. Alisertib also induced cell cycle arrest. The treatment of ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of alisertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We investigated the alisertib activity against T315I positive cells. Alisertib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation and induced cell cycle arrest. We investigated the efficacy between ponatinib and alisertib by using these cell lines. Combined treatment of Ba/F3 T315I cells with ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Ponatinib and alisertib were also effective against T315I primary samples. We examined the intracellular signaling of alisertib. Phosphorylation of Aurora A was inhibited in a time dependent manner. We also found the phosphorylation of histone H3 was also reduced in a dose dependent manner suggested that high concentration of alisertib also inhibits Aurora B activity. We next investigated by using ponatinib resistant Ba/F3 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of alisertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells in the dose dependent manner. Alisertib induced cell cycle arrest in ponatinib resistant cells. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and alisertib caused significantly more cytotoxicity. To assess the activity of alisertib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with 1×107 Ba/F3 T315I cells. A dose of 30 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of alisertib combination strategies in the clinic. Data from this study suggested that administration of the ponatinib and Aurora inhibitor, alisertib may be a powerful strategy against BCR-ABL mutant cells including T315I. Disclosures: No relevant conflicts of interest to declare.
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Metzger, Eric, Na Yin, Melanie Wissmann, Natalia Kunowska, Kristin Fischer, Nicolaus Friedrichs, Debasis Patnaik, et al. "Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation." Nature Cell Biology 10, no. 1 (December 9, 2007): 53–60. http://dx.doi.org/10.1038/ncb1668.
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Tan, Ee Phie, Sarah Caro, Anish Potnis, Christopher Lanza, and Chad Slawson. "O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization." Journal of Biological Chemistry 288, no. 38 (August 14, 2013): 27085–99. http://dx.doi.org/10.1074/jbc.m113.470187.
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Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.
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Niedzialkowska, Ewa, Fangwei Wang, Przemyslaw J. Porebski, Wladek Minor, Jonathan M. G. Higgins, and P. Todd Stukenberg. "Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres." Molecular Biology of the Cell 23, no. 8 (April 15, 2012): 1457–66. http://dx.doi.org/10.1091/mbc.e11-11-0904.
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Survivin, a subunit of the chromosome passenger complex (CPC), binds the N-terminal tail of histone H3, which is phosphorylated on T3 by Haspin kinase, and localizes the complex to the inner centromeres. We used x-ray crystallography to determine the residues of Survivin that are important in binding phosphomodified histone H3. Mutation of amino acids that interact with the histone N-terminus lowered in vitro tail binding affinity and reduced CPC recruitment to the inner centromere in cells, validating our solved structures. Phylogenetic analysis shows that nonmammalian vertebrates have two Survivin paralogues, which we name class A and B. A distinguishing feature of these paralogues is an H-to-R change in an amino acid that interacts with the histone T3 phosphate. The binding to histone tails of the human class A paralogue, which has a histidine at this position, is sensitive to changes around physiological pH, whereas Xenopus Survivin class B is less so. Our data demonstrate that Survivin paralogues have different characteristics of phosphospecific binding to threonine-3 of histone H3, providing new insight into the biology of the inner centromere.
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Dastidar, Eeshita G., Kristina Dzeyk, Jeroen Krijgsveld, Nicholas A. Malmquist, Christian Doerig, Artur Scherf, and Jose-Juan Lopez-Rubio. "Comprehensive Histone Phosphorylation Analysis and Identification of Pf14-3-3 Protein as a Histone H3 Phosphorylation Reader in Malaria Parasites." PLoS ONE 8, no. 1 (January 7, 2013): e53179. http://dx.doi.org/10.1371/journal.pone.0053179.
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Parwani, Kiran, Jennifer Spangle, Leon McSwain, Ramona Haji Seyed Javadi, Anna Kenney, and David Yu. "DNAR-06. LOSS OF HISTONE H3 THREONINE 45 PHOSPHORYLATION DECREASES H3K36ME3 TO ABROGATE THE RADIATION-INDUCED DNA DAMAGE IN GLIOBLASTOMA MULTIFORME." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii91. http://dx.doi.org/10.1093/neuonc/noac209.338.
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Abstract Glioblastoma multiforme (GBM) is the most common and fatal adult brain tumour. As radioresistance is a common occurrence and contributes to poor patient prognosis, exploiting the mechanistic basis of GBM radioresistance to develop new therapeutic approaches remains an unmet clinical need. Chromatin post-translational modifications (PTMs) can augment cancer radioresistance. Several chromatin PTMs are regulated by PI3K/AKT, which is frequently hyperactivated in GBM. Previous research demonstrated that the PI3K effector AKT phosphorylates histone H3 at threonine 45 (pH3T45) in response to DNA damage. To genetically dissect the function of H3T45 phosphorylation in the DNA damage response, we engineered the PI3K pathway-activated U87MG GBM cell line to stably overexpress wildtype H3, or a phospho-null H3T45A mutant. We show that in both the presence and absence of irradiation, cells harbouring the phospho-null H3T45A mutant decreases H3K36me3 on free and nucleosome-incorporated histones, which may suggest a deficiency in DNA repair protein recruitment. By surveying irradiation-induced 53BP1 foci formation, we demonstrate that loss of pH3T45 compromises damage repair through NHEJ by delaying the onset of repair signaling. We also observe a defect in DNA damage resolution, which enhances apoptosis to reduce cell clonogenicity and proliferative capacity. Irradiation does not further reduce clonogenicity and proliferation in the phospho-null H3T45A-expressing cells. Taken together, these data suggest a critical function for PI3K-directed H3T45 phosphorylation in DNA damage repair and that loss of pH3T45 may contribute to GBM radioresistance. We anticipate that further characterization of this phosphorylation event will inform the understanding of GBM radioresistance and may suggest novel therapeutic strategies.
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Espinos, Estelle, Agathe Le Van Thaï, Christelle Pomiès, and Michel J. Weber. "Cooperation between Phosphorylation and Acetylation Processes in Transcriptional Control." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3474–84. http://dx.doi.org/10.1128/mcb.19.5.3474.
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ABSTRACT We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118–124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 μM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H10 gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148–24153, 1997) showed that the induction of the H10 gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.
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Harrison, J. G., and A. Clerk. "Phosphorylation of Histone 3 (H3) in cardiac myocytes subjected to hyperosmotic shock." Biochemical Society Transactions 28, no. 5 (October 1, 2000): A429. http://dx.doi.org/10.1042/bst028a429a.
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Weissmann, Frank, Inhua Muyrers-Chen, Tanja Musch, Dirk Stach, Manfred Wiessler, Renato Paro, and Frank Lyko. "DNA Hypermethylation in Drosophila melanogaster Causes Irregular Chromosome Condensation and Dysregulation of Epigenetic Histone Modifications." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2577–86. http://dx.doi.org/10.1128/mcb.23.7.2577-2586.2003.
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ABSTRACT The level of genomic DNA methylation plays an important role in development and disease. In order to establish an experimental system for the functional analysis of genome-wide hypermethylation, we overexpressed the mouse de novo methyltransferase Dnmt3a in Drosophila melanogaster. These flies showed severe developmental defects that could be linked to reduced rates of cell cycle progression and irregular chromosome condensation. In addition, hypermethylated chromosomes revealed elevated rates of histone H3-K9 methylation and a more restricted pattern of H3-S10 phosphorylation. The developmental and chromosomal defects induced by DNA hypermethylation could be rescued by mutant alleles of the histone H3-K9 methyltransferase gene Su(var)3-9. This mutation also resulted in a significantly decreased level of genomic DNA methylation. Our results thus uncover the molecular consequences of genomic hypermethylation and demonstrate a mutual interaction between DNA methylation and histone methylation.
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Smith, James A. H., Tertius A. Kohn, Ashley K. Chetty, and Edward O. Ojuka. "CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene." American Journal of Physiology-Endocrinology and Metabolism 295, no. 3 (September 2008): E698—E704. http://dx.doi.org/10.1152/ajpendo.00747.2007.
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The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 × 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased ∼2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.
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Kelly, Alexander E., Cristina Ghenoiu, John Z. Xue, Christian Zierhut, Hiroshi Kimura, and Hironori Funabiki. "Survivin Reads Phosphorylated Histone H3 Threonine 3 to Activate the Mitotic Kinase Aurora B." Science 330, no. 6001 (August 12, 2010): 235–39. http://dx.doi.org/10.1126/science.1189505.
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Chandrasekaran, V., G. Katona, M. I. Bokarewa, K. M. Andersson, M. C. Erlandsson, M. Jensen, N. Oparina, and A. Damdimopoulos. "POS0397 AGGREGATED SURVIVIN BINDING AROUND HISTONE H3 EPIGENETIC MODIFICATIONS IN RISK LOCI ASSOCIATED WITH RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 428.1–428. http://dx.doi.org/10.1136/annrheumdis-2021-eular.3212.
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Background:Survivin is an integral part of the Chromosomal Passenger Complex (CPC) which plays a vital role in mitosis. Experiments have demonstrated that survivin can physically bind to DNA. Crystallographic studies show that survivin binds to Threonine-3 of histone H3. In patients with autoimmune diseases, increased survivin expression contributes to an aggravated disease phenotype. Thus, functional, and mechanistic data point to a potential chromatin regulatory role for survivin, possibly in combination with the established gene regulatory function carried out by histone epigenetic modifications (EM).Objectives:The objective of the study was to analyse the co-localization of chromatin bound survivin with three histone H3 epigenetic modifications – acetylated lysine 27 (K27ac) and trimethylated lysine 4 (K4me3) and lysine-27 (K27me3). The second objective was to analyse if survivin-bound DNA sequences overlapped with sequences in the vicinity of 106 GWAS SNPs that are associated with a risk of developing rheumatoid arthritis (RA).Methods:Chromatin from CD4 T cells of 14 female subjects was immunoprecipitated with survivin antibodies and each of the histone H3 antibodies, and coupled with sequencing (ChIPseq, Hiseq2000, Illumina). After mapping the annotations of sequenced regions to the human reference genome hg38, enriched peaks were identified through Homer software. The identified survivin ChIP peaks were analysed for colocalization with peaks of the three histone H3 EMs and with RA risk loci, using the Bioconductor package ‘ChIPPeakAnno’ through RStudio.Results:Among the total of ~13,000 individual survivin ChIP-peaks, 33% colocalized with histone H3 EM peaks. The overlapping peaks show a linear increase in average peak size compared with the peaks showing no colocalization with any H3 EM peak. A maximum of 5.5-fold increase in average peak size was observed when survivin bound peaks overlap with peaks of all three H3 EMs. A major proportion (86%) of top RA risk SNPs was associated with either binding of survivin or H3 EMs. In this subset, 63% of RA risk SNPs were found within an area of 100 kilobases from survivin ChIP-peaks, with preferential enrichment of high-scoring peaks when survivin colocalizes with all 3 H3 EMs. Survivin was bound to risk SNPs annotated to, among others, the major immunological genes CD83, IRF4, CD28, ICOS and IL2RAConclusion:This study presents experimental evidence that survivin binding to DNA preferentially occurred in regions with high density of histone EMs. The increased aggregation of survivin around histone H3 EMs point to its potential regulatory function in gene transcription. Since regions around RA risk SNPs overlap with survivin peaks, survivin’s nuclear function could have immunologically important effects in mechanisms of autoimmune diseases.Disclosure of Interests:None declared
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Pancholi, Vijaykumar, and Vincent A. Fischetti. "Regulation of the Phosphorylation of Human Pharyngeal Cell Proteins by Group A Streptococcal Surface Dehydrogenase: Signal Transduction between Streptococci and Pharyngeal Cells." Journal of Experimental Medicine 186, no. 10 (November 17, 1997): 1633–43. http://dx.doi.org/10.1084/jem.186.10.1633.
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Whether cell-to-cell communication results when group A streptococci interact with their target cells is unknown. Here, we report that upon contact with cultured human pharyngeal cells, both whole streptococci and purified streptococcal surface dehydrogenase (SDH) activate pharyngeal cell protein tyrosine kinase as well as protein kinase C, thus regulating the phosphorylation of cellular proteins. SDH, a major surface protein of group A streptococci, has both glyceraldehyde-3-phosphate dehydrogenase and ADP-ribosylating enzyme activities that may relate to early stages of streptococcal infection. Intact streptococci and purified SDH induce a similar protein phosphorylation pattern with the de novo tyrosine phosphorylation of a 17-kD protein found in the membrane/particulate fraction of the pharyngeal cells. However, this phosphorylation required the presence of cytosolic components. NH2-terminal amino acid sequence analysis identified the 17-kD protein as nuclear core histone H3. Both phosphotyrosine and phosphoserine-specific monoclonal antibodies reacted with the 17-kD protein by Western blot, suggesting that the binding of SDH to these pharyngeal cells elicits a novel signaling pathway that ultimately leads to activation of histone H3–specific kinases. Genistein-inhibitable phosphorylation of histone H3 indicates that tyrosine kinase plays a key role in this event. Treatment of pharyngeal cells with protein kinase inhibitors such as genistein and staurosporine significantly inhibited streptococcal invasion of pharyngeal cells. Therefore, these data indicated that streptococci/SDH-mediated phosphorylation plays a critical role in bacterial entry into the host cell. To identify the membrane receptor that elicits these signaling events, we found that SDH bound specifically to 30- and 32-kD membrane proteins in a direct ligand-binding assay. These findings clearly suggest that SDH plays an important role in cellular communication between streptococci and pharyngeal cells that may be important in host cell gene transcription, and hence in the pathogenesis of streptococcal infection.
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Lucero, Héctor A., Néstor Cortez, and Rubén H. Vallejos. "Light modulation of serine and threonine phosphorylation in histone III by thylakoids." Biochimica et Biophysica Acta (BBA) - Bioenergetics 890, no. 1 (January 1987): 77–81. http://dx.doi.org/10.1016/0005-2728(87)90070-3.
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Happel, Nicole, Stefan Stoldt, Bernhard Schmidt, and Detlef Doenecke. "M Phase-Specific Phosphorylation of Histone H1.5 at Threonine 10 by GSK-3." Journal of Molecular Biology 386, no. 2 (February 2009): 339–50. http://dx.doi.org/10.1016/j.jmb.2008.12.047.
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Chandrasekaran, V., M. I. Bokarewa, N. Oparina, K. M. Andersson, G. Katona, M. Erlandsson, M. Jensen, and A. Damdimopoulos. "POS0032 FUNCTIONAL ROLE OF SURVIVIN IN ORGANIZATION OF BIVALENT CHROMATIN REGIONS AND CONSEQUENCE FOR ARTHRITIS-RELEVANT GENE EXPRESSION." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 231.3–231. http://dx.doi.org/10.1136/annrheumdis-2022-eular.4856.
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BackgroundBivalent chromatin (BvCR) is characterized by the presence of simultaneous active and repressive modifications on histone H3 proteins. Influencing expression of the genes, BvCR determine cell fate and direct differentiation and lineage commitment in primary T cells and contribute to autoimmunity. Survivin is highly expressed during cell division and in effector Th1 cells contributing to aggravation of autoimmune inflammation. Survivin can physically bind to DNA, specifically to Threonine-3 of histone H3 (1). Thus, functional, and mechanistic data point to a potential chromatin regulatory role for survivin, potentially acting in combination with histone epigenetic modifications (EMs).ObjectivesThe goal of our study is to establish the colocalization of survivin with BvCRs and to deduce functional effects of this collaboration on chromatin organization and gene expression.MethodsChromatin from CD4+ T cells of 14 female subjects was immunoprecipitated with survivin antibodies and histone H3K27ac, H3K27me3, H3K4me3 antibodies, and coupled with DNA sequencing (ChIPseq, Hiseq2000, Illumina). BvCR were identified as exact overlaps of the three histone EM peaks and the overlapping regions were searched for co-localization with survivin using the ‘ChIPPeakAnno’ Bioconductor package. Tag counts K27me3>K27ac were defined as inactive/poised BvCR, while tag count K27me3<K27ac were identified asactive BvCR. Motif search was done through the MEME tool, and high/moderate complexity motifs with E-value >10e-5 were selected and scanned through the HOCOMOCO database to identify consensus transcription factor (TF) motifs. TFs co-localized with the BvCD were identified through ReMap database. To identify survivin sensitive genes, CD4+ T cells were treated with survivin inhibitor YM155 and a list of reproducible DEG (log2FC>[0.4], >1 experiment) was mapped and analysed for clustering with BvCR.ResultsCo-localization of survivin ChIP peaks with individual H3-peaks was significantly less frequent compared to overlap with all three (a3)-H3 BvCR (7.1 vs 29.8%, p=8.9e-13). Overlap of a3-H3 peaks not containing survivin was less frequent (34%) compared to those which contained survivin (66%). Notably, survivin peak size was 5.5-fold higher when colocalized with a3-H3 peaks, compared to no, or any single H3 (p<2.2e-16). In contrast, no size difference for any of the H3 EM peaks was found.Further analysis of two non-redundant groups of BvCR that contain (survivin-a3H3, n=4085), and not containing survivin (a3H3noSurv, n = 2131) demonstrated that survivin was mostly associated with inactive BvCR (OR1.29, p=6.6e-6), while no such specificity was found for BvCR with no survivin. Additionally, survivin containing BvCR contained abundant binding sites matching known consensus TF motifs. No sequence-specific motifs were identified in BvCR with no survivin. Comparison of results obtained through HOCOMOCO and ReMap databases resulted in a list of 68 unique TFs. Many of those are key regulators of adaptive immune responses, cellular metabolism, and pluripotency. Differentially expressed genes mapped to BvCR demonstrated enrichment for cellular hormone metabolic processes, regeneration and DNA biosynthesis.ConclusionThis study provides experimental evidence that survivin defines binding specificity in bivalent chromatin regions being associated with regulation of cellular metabolism and renewal of CD4+ T cells that are functionally important to resist autoimmunity.References[1]Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B Science. 2010 Oct 8; 330(6001): 235–239.Disclosure of InterestsNone declared
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Feizbakhsh, Omid, Florian Pontheaux, Virginie Glippa, Julia Morales, Sandrine Ruchaud, Patrick Cormier, and Fernando Roch. "A Peak of H3T3 Phosphorylation Occurs in Synchrony with Mitosis in Sea Urchin Early Embryos." Cells 9, no. 4 (April 7, 2020): 898. http://dx.doi.org/10.3390/cells9040898.
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The sea urchin embryo provides a valuable system to analyse the molecular mechanisms orchestrating cell cycle progression and mitosis in a developmental context. However, although it is known that the regulation of histone activity by post-translational modification plays an important role during cell division, the dynamics and the impact of these modifications have not been characterised in detail in a developing embryo. Using different immuno-detection techniques, we show that the levels of Histone 3 phosphorylation at Threonine 3 oscillate in synchrony with mitosis in Sphaerechinus granularis early embryos. We present, in addition, the results of a pharmacological study aimed at analysing the role of this key histone post-translational modification during sea urchin early development.
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Li, Ji, Peili Chen, Natasha Sinogeeva, Myriam Gorospe, Robert P. Wersto, Francis J. Chrest, Janice Barnes, and Yusen Liu. "Arsenic Trioxide Promotes Histone H3 Phosphoacetylation at the Chromatin ofCASPASE-10in Acute Promyelocytic Leukemia Cells." Journal of Biological Chemistry 277, no. 51 (October 17, 2002): 49504–10. http://dx.doi.org/10.1074/jbc.m207836200.
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Arsenic trioxide (As2O3) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As2O3is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As2O3in acute promyelocytic leukemia NB4 cells.CASPASE-10expression was found to be potently induced by As2O3. Accordingly, caspase-10 activity also substantially increased in response to As2O3treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As2O3-triggered apoptosis. Interestingly, the treatment of NB4 cells with As2O3markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As2O3potently enhances histone H3 phosphoacetylation at theCASPASE-10locus. These results suggest that the effect of As2O3on histone H3 phosphoacetylation at theCASPASE-10gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.