Journal articles on the topic 'Conflicts between DNA replication and transcription'
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Saponaro, Marco. "Transcription–Replication Coordination." Life 12, no. 1 (January 13, 2022): 108. http://dx.doi.org/10.3390/life12010108.
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Transcription and replication are the two most essential processes that a cell does with its DNA: they allow cells to express the genomic content that is required for their functions and to create a perfect copy of this genomic information to pass on to the daughter cells. Nevertheless, these two processes are in a constant ambivalent relationship. When transcription and replication occupy the same regions, there is the possibility of conflicts between transcription and replication as transcription can impair DNA replication progression leading to increased DNA damage. Nevertheless, DNA replication origins are preferentially located in open chromatin next to actively transcribed regions, meaning that the possibility of conflicts is potentially an accepted incident for cells. Data in the literature point both towards the existence or not of coordination between these two processes to avoid the danger of collisions. Several reviews have been published on transcription–replication conflicts, but we focus here on the most recent findings that relate to how these two processes are coordinated in eukaryotes, considering advantages and disadvantages from coordination, how likely conflicts are at any given time, and which are their potential hotspots in the genome.
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Million-Weaver, Samuel, Ariana Nakta Samadpour, and Houra Merrikh. "Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis." Journal of Bacteriology 197, no. 14 (May 4, 2015): 2374–82. http://dx.doi.org/10.1128/jb.00237-15.
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ABSTRACTEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription inBacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.IMPORTANCEHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms forBacillus subtiliscells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.
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RUDOLPH, C., P. DHILLON, T. MOORE, and R. LLOYD. "Avoiding and resolving conflicts between DNA replication and transcription." DNA Repair 6, no. 7 (July 1, 2007): 981–93. http://dx.doi.org/10.1016/j.dnarep.2007.02.017.
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Urban, Vaclav, Jana Dobrovolna, Daniela Hühn, Jana Fryzelkova, Jiri Bartek, and Pavel Janscak. "RECQ5 helicase promotes resolution of conflicts between replication and transcription in human cells." Journal of Cell Biology 214, no. 4 (August 8, 2016): 401–15. http://dx.doi.org/10.1083/jcb.201507099.
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Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here, we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome-stabilizing effect by acting at sites of replication-transcription collisions. Moreover, RECQ5-deficient cells accumulate RAD18 foci and BRCA1-dependent RAD51 foci that are both formed at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Finally, we provide evidence for a novel mechanism of resolution of replication-transcription collisions wherein the interaction between RECQ5 and proliferating cell nuclear antigen (PCNA) promotes RAD18-dependent PCNA ubiquitination and the helicase activity of RECQ5 promotes the processing of replication intermediates.
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Lang, Kevin S., and Houra Merrikh. "The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria." Annual Review of Microbiology 72, no. 1 (September 8, 2018): 71–88. http://dx.doi.org/10.1146/annurev-micro-090817-062514.
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Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.
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Tehranchi, Ashley K., Matthew D. Blankschien, Yan Zhang, Jennifer A. Halliday, Anjana Srivatsan, Jia Peng, Christophe Herman, and Jue D. Wang. "The Transcription Factor DksA Prevents Conflicts between DNA Replication and Transcription Machinery." Cell 141, no. 4 (May 2010): 595–605. http://dx.doi.org/10.1016/j.cell.2010.03.036.
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McGlynn, Peter, Nigel J. Savery, and Mark S. Dillingham. "The conflict between DNA replication and transcription." Molecular Microbiology 85, no. 1 (May 31, 2012): 12–20. http://dx.doi.org/10.1111/j.1365-2958.2012.08102.x.
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St Germain, Commodore P., Hongchang Zhao, Vrishti Sinha, Lionel A. Sanz, Frédéric Chédin, and Jacqueline H. Barlow. "Genomic patterns of transcription–replication interactions in mouse primary B cells." Nucleic Acids Research 50, no. 4 (January 31, 2022): 2051–73. http://dx.doi.org/10.1093/nar/gkac035.
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Abstract Conflicts between transcription and replication machinery are a potent source of replication stress and genome instability; however, no technique currently exists to identify endogenous genomic locations prone to transcription–replication interactions. Here, we report a novel method to identify genomic loci prone to transcription–replication interactions termed transcription–replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1009 unique transcription–replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. TRIs are highly enriched at transcription start sites and map to early replicating regions. TRIs exhibit enhanced Replication Protein A association and TRI-associated genes exhibit higher replication fork termination than control transcription start sites, two marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.
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Trautinger, Brigitte W., Razieh P. Jaktaji, Ekaterina Rusakova, and Robert G. Lloyd. "RNA Polymerase Modulators and DNA Repair Activities Resolve Conflicts between DNA Replication and Transcription." Molecular Cell 19, no. 2 (July 2005): 247–58. http://dx.doi.org/10.1016/j.molcel.2005.06.004.
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Stevenson-Jones, Flint, Jason Woodgate, Daniel Castro-Roa, and Nikolay Zenkin. "Ribosome reactivates transcription by physically pushing RNA polymerase out of transcription arrest." Proceedings of the National Academy of Sciences 117, no. 15 (April 1, 2020): 8462–67. http://dx.doi.org/10.1073/pnas.1919985117.
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In bacteria, the first two steps of gene expression—transcription and translation—are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription–translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.
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Shao, Xin, Amalie M. Joergensen, Niall G. Howlett, Michael Lisby, and Vibe H. Oestergaard. "A distinct role for recombination repair factors in an early cellular response to transcription–replication conflicts." Nucleic Acids Research 48, no. 10 (April 24, 2020): 5467–84. http://dx.doi.org/10.1093/nar/gkaa268.
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Abstract Transcription–replication (T–R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T–R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T–R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T–R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.
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Marabitti, Veronica, Pasquale Valenzisi, Giorgia Lillo, Eva Malacaria, Valentina Palermo, Pietro Pichierri, and Annapaola Franchitto. "R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1." International Journal of Molecular Sciences 23, no. 3 (January 28, 2022): 1547. http://dx.doi.org/10.3390/ijms23031547.
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Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA–RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.
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Colizzi, Enrico Sandro, and Paulien Hogeweg. "Transcriptional Mutagenesis Prevents Ribosomal DNA Deterioration: The Role of Duplications and Deletions." Genome Biology and Evolution 11, no. 11 (October 25, 2019): 3207–17. http://dx.doi.org/10.1093/gbe/evz235.
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Abstract Clashes between transcription and replication complexes can cause point mutations and chromosome rearrangements on heavily transcribed genes. In eukaryotic ribosomal RNA genes, the system that prevents transcription–replication conflicts also causes frequent copy number variation. Such fast mutational dynamics do not alter growth rates in yeast and are thus selectively near neutral. It was recently found that yeast regulates these mutations by means of a signaling cascade that depends on the availability of nutrients. Here, we investigate the long-term evolutionary effect of the mutational dynamics observed in yeast. We developed an in silico model of single-cell organisms whose genomes mutate more frequently when transcriptional load is larger. We show that mutations induced by high transcriptional load are beneficial when biased toward gene duplications and deletions: they decrease mutational load even though they increase the overall mutation rates. In contrast, genome stability is compromised when mutations are not biased toward gene duplications and deletions, even when mutations occur much less frequently. Taken together, our results show that the mutational dynamics observed in yeast are beneficial for the long-term stability of the genome and pave the way for a theory of evolution where genetic operators are themselves cause and outcome of the evolutionary dynamics.
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Dutrieux, Laure, Yea-Lih Lin, Malik Lutzmann, Guilhem Requirand, Nicolas Robert, Laure Vincent, Guillaume Cartron, et al. "Exploiting Transcription-Replication Conflicts As a Novel Therapeutic Intervention in Multiple Myeloma." Blood 138, Supplement 1 (November 5, 2021): 1582. http://dx.doi.org/10.1182/blood-2021-151530.
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Abstract Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained in malignant PCs meaning that these cells display an elevated transcriptional stress. Besides, malignant PCs face oncogene-induced replication stress concomitantly with cell cycle deregulation. Consequently, transcription and replication in malignant PCs need to be tightly coordinated to avoid too much interferences that would increase replication stress and genomic instability. A failure to cope with these transcription/replication conflicts (TRCs) could have a significant impact on mutagenesis involved in MM development. Importantly, these effects might open the therapeutic possibility of TRCs enhancement to specifically kill malignant PCs. Based on these observations, we identified a signature of 13 TRCs resolution factors significantly overexpressed in MM patients. Considering the potent role of TRCs resolution in MM cell adaptation to replication stress, we sought to identify the TRCs resolution factors that are associated with a poor outcome in MM. High expression of 9 out of the 13 TRCs resolution factors significantly overexpressed in malignant PCs are associated with a poor outcome in MM (TT2 cohort, n = 345). We gathered the prognostic value of these 9 genes within a Gene Expression Profile (GEP)-based TRC resolution score (TRC score). High TRC score is associated with a poor outcome in two independent cohorts of newly diagnosed MM patients treated by high dose therapy and autologous stem cell transplantation (Arkansas, TT2 cohort, n = 345; CoMMpass cohort, n = 674) (Fig.1A). Interestingly, we investigated the link between the TRC score and the MM cells drug response using our collection of human myeloma cell lines (HMCLs), and identified that HMCLs with high TRC score values are significantly more sensitive to Panobinostat histone deacetylase inhibitor, currently used in MM treatment at relapse (n = 11, p value < 0.05). Histone acetylation has been shown to promote R-loop formation that constitutes obstacles to replication fork progression. Using primary MM cells from patients (n = 12) co-cultured with their bone marrow microenvironment, we found that a high TRC score value is associated with a higher toxicity of Panobinostat (p value < 0.01). Therefore, the TRC score allows the identification of a MM patients subgroup with a poor outcome that could benefit from Panobinostat treatment. Interestingly, TRCs are promoted by R-loop formation and G-quadruplex (G4) stabilizers treatment. R-loops are formed by the reannealing of the nascent RNA with the template DNA (called an RNA:DNA hybrid). G4s are four-stranded secondary DNA structures, constituted of stacked guanine tetrads. Both structures are formed during transcription in G-rich DNA regions and can represent a barrier for replication fork progression if unscheduled. G4s can stabilize R-loops which have been shown to mediate DNA damage induced by G4 stabilizers. Interestingly, treatment with the G4 stabilizer Pyridostatin (PDS) was associated with significant toxicity on HMCLs (n = 15) (Fig.1B), and on primary MM cells of patients cocultured with their bone marrow microenvironment (n = 5, p value < 0.05). Interestingly, the combination of PDS and Panobinostat has a synergistic effect in HMCLs. We also found a correlation between HMCLs TRC score and the response to two Bromodomain and Extra-Terminal motif (BET) proteins inhibitors, I-BET-762 and RVX-208. The synergistic effect of PDS combination with I-BET-762 was validated in vitro. BET proteins inhibition has been shown to increase R-loop formation and DNA damage. Furthermore, we used inducible RNase H expression in HMCLs to specifically degrade RNA:DNA hybrids. RNase H expression resulted in a significant reduction of DNA damage response after PDS treatment (Fig.1C). Our results underline that spontaneous replication stress and genomic instability are related to R-loop formation and TRCs in MM cells. Altogether, these results emphasize the therapeutic potential of TRCs targeting in MM using G4 stabilizers alone or in combination with current treatments. Figure 1 Figure 1. Disclosures Vincent: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Cartron: Roche, Celgene-BMS: Consultancy; Danofi, Gilead, Novartis, Jansen, Roche, Celgene-BMS, Abbvie, Takeda: Honoraria. Herbaux: Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Moreaux: Diag2Tec: Consultancy.
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Moriel-Carretero, María, Sara Ovejero, Marie Gérus-Durand, Dimos Vryzas, and Angelos Constantinou. "Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors." Journal of Cell Biology 216, no. 12 (October 13, 2017): 4007–26. http://dx.doi.org/10.1083/jcb.201702136.
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Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes.
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Nickoloff, Jac A., Neelam Sharma, Lynn Taylor, Sage J. Allen, and Robert Hromas. "Nucleases and Co-Factors in DNA Replication Stress Responses." DNA 2, no. 1 (March 1, 2022): 68–85. http://dx.doi.org/10.3390/dna2010006.
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DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics.
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Halazonetis, Thanos D., Michalis Petropoulos, Giacomo G. Rossetti, Angeliki Karamichali, Alena Freudenmann, Luca Iacovino, Vasilis Dionellis, and Sotirios K. Sotiriou. "Abstract 1566: DNA damage generated by transcription-replication conflicts explains the synthetic lethality of PARP inhibitors with homologous recombination deficiency." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1566. http://dx.doi.org/10.1158/1538-7445.am2023-1566.
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Abstract An important advance in cancer therapy in the last decade has been the development of poly ADP-ribose polymerase (PARP) inhibitors for the treatment of select ovarian and breast cancers. The clinical benefit stems from the synthetic lethality of PARP inhibitors with homologous recombination (HR) deficiency, which deficiency is prevalent in the cancer types listed above. The current model to explain the synthetic lethality is based on the observation that PARP inhibitors trap PARPs on DNA: the trapped PARPs block progression of the replisome, leading to the formation of DNA double-strand breaks (DSBs), which require HR for repair. Here, we propose a novel mechanism to explain the synthetic lethality between PARP inhibitors and HR deficiencies. We show that PARP1 functions together with the proteins TIMELESS and TIPIN to protect the replisome from transcription-replication conflicts (TRCs). In the absence of any one of these proteins, TRCs evolved into DNA DSBs that required HR for repair, explaining the observed synthetic lethality. In further support of this model, when we inhibited transcription elongation, which prevents the emergence of transcription-replication conflicts, then the HR-deficient cancer cells became resistant to PARP inhibitors. We further observed that trapping of PARPs on DNA was not required for the synthetic lethality with HR deficiency, since we could observe strong synthetic lethality simply by depleting PARP1 and PARP2 by siRNA. Rather, trapping of PARPs on DNA correlated with the ability of the various PARP inhibitors to inhibit PARP enzymatic activity in cells; the strongest trappers were also the most potent inhibitors of PARP1 in cells. In vitro, all PARP inhibitors tested were almost equipotent in their ability to inhibit the enzymatic activity of PARP1, meaning that the potency of PARP1 inhibitors in vitro did not reflect their inhibitory potency in cells. Our model provides a new framework for understanding the mechanism of action of PARP inhibitors in the clinic and the mechanisms by which resistance can emerge. Citation Format: Thanos D. Halazonetis, Michalis Petropoulos, Giacomo G. Rossetti, Angeliki Karamichali, Alena Freudenmann, Luca Iacovino, Vasilis Dionellis, Sotirios K. Sotiriou. DNA damage generated by transcription-replication conflicts explains the synthetic lethality of PARP inhibitors with homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1566.
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Redington, Jennifer, Jaigeeth Deveryshetty, Lakshmi Kanikkannan, Ian Miller, and Sergey Korolev. "Structural Insight into the Mechanism of PALB2 Interaction with MRG15." Genes 12, no. 12 (December 17, 2021): 2002. http://dx.doi.org/10.3390/genes12122002.
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The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.
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Rimmelé, Pauline, Jean-Hugues Guervilly, Filippo Rosselli, Françoise Moreau-Gachelin, and Christel Guillouf. "Spi-1/PU.1 Accelerates Replication Fork Elongation through PP1a Phosphatase-Associated Dephosphorylation of CHK1 in Erythroleukemic Cells." Blood 124, no. 21 (December 6, 2014): 2193. http://dx.doi.org/10.1182/blood.v124.21.2193.2193.
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Abstract The multistage process of leukemic formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Oncogenes and tumor suppressors trigger a replicative stress, that will consequently participates to the progression of cancer partly through this increased genetic instability. Thus, determining how cells cope with replicative stress should help understanding leukemogenesis and lead to the identification of new targets for antitumor treatments. Using a multistep leukemia model driven by Spi-1/PU.1 overexpression, we investigated the relationship between DNA replication and cancer progression. We have previously identified that the constitutive overexpression of the oncogenic transcription factor Spi-1/PU.1 is associated with an increased speed of DNA chain elongation favoring genetic instability without inducing DNA strand breaks. The Spi-1-induced replicative stress is peculiar because, in contrast to most of stress triggered by oncogenes or tumor suppressor, it is associated with an increase fork progression speed instead of alteration in the program of origin firing. Here, we bring evidence that the S phase checkpoint protein, CHK1, is maintained in the inactive dephosphorylated form in Spi-1/PU.1 overexpressing pre-leukemic cells inducing and/or maintaining the observed high speed of DNA chain elongation. CHK1 under-phosphorylation is not due to defects in ATR signaling, its main regulator. Moreover, pharmacological inhibition of the kinases, ATM and DNA-PK, did not decrease CHK1 phosphorylation in the preleukemic cells overexpressing Spi-1. These findings are not consistent with an involvement of DNA damage response kinases in Spi-1-mediated modulation of CHK1 phosphorylation.We found that PP1a expression is increased in Spi-1/PU.1 overexpressing pre-leukemic cells compared to cells in which Spi-1/PU.1 was down-regulated. Two functional assays bring arguments that PP1 activity mediates the Spi-1/PU.1 effect on CHK1 dephosphorylation. Indeed, inhibition of PP1activity in cells overexpressing Spi-1 promoted the phosphorylation of CHK1, while the overexpression of PP1a led to the loss of a correlation between CHK1 phosphorylation and Spi-1 expression. In addition, PP1a inhibition and overexpression, not only acted on the CHK1 phosphorylation status controlled by Spi-1 but also inversely modified the progression of replication. Altogether, these results support the existence of a pathway linking Spi-1/PU.1 expression to acceleration of DNA replication via a PP1-mediated control of CHK1 phosphorylation in the pre-leukemic cells. These results identified a new pathway by which an oncogene influences replicative stress and favors the leukemic progression by fostering the incidence of genomic instability. Disclosures No relevant conflicts of interest to declare.
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Uruci, Sidrit, Calvin Shun Yu Lo, David Wheeler, and Nitika Taneja. "R-Loops and Its Chro-Mates: The Strange Case of Dr. Jekyll and Mr. Hyde." International Journal of Molecular Sciences 22, no. 16 (August 17, 2021): 8850. http://dx.doi.org/10.3390/ijms22168850.
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Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.
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Shinnick, Kathryn M., Kelly A. Barry, Elizabeth A. Eklund, and Thomas J. McGarry. "Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation." Blood 114, no. 22 (November 20, 2009): 1478. http://dx.doi.org/10.1182/blood.v114.22.1478.1478.
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Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.
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Hoffman, Elizabeth A., Andrew McCulley, Brian Haarer, Remigiusz Arnak, and Wenyi Feng. "Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription." Genome Research 25, no. 3 (January 21, 2015): 402–12. http://dx.doi.org/10.1101/gr.180497.114.
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Agarwal, Poonam, and Kyle M. Miller. "The nucleosome: orchestrating DNA damage signaling and repair within chromatin." Biochemistry and Cell Biology 94, no. 5 (October 2016): 381–95. http://dx.doi.org/10.1139/bcb-2016-0017.
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DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.
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Chen, Zhe, Lei Li, Jieping Chen, and Yu Hou. "Nuclear Protein DEK Governs Quiescence and Metabolic Homeostasis of Hematopoietic Stem Cells By Shaping Chromatin Accessibility." Blood 136, Supplement 1 (November 5, 2020): 7. http://dx.doi.org/10.1182/blood-2020-136859.
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Hematopoietic stem cells (HSCs) must achieve a balance between quiescence and activation that fulfils the demands for hematopoiesis without compromising long-term maintenance of HSCs. DEK, a chromatin architectural factor, is involved in chromatin remodeling, transcriptional regulation and DNA replication, and is implicated in genetic and epigenetic regulation of gene expression. Here, we identified that DEK is a critical regulator of HSCs quiescence. Deletion of DEK in mice resulted in abnormal hematopoiesis with an obvious decreased HSC pool size (~3700 to ~1700 cells/mouse), associated with apparent reduction in the proportion of HSCs in G0 phase as compared to control HSCs (~72% to ~57%). As shown by serial bone marrow transplantation and competitive repopulation assays, deletion of DEK impaired the self-renewal capacity of HSCs. Mechanistically, deficiency of DEK in HSC altered chromatin accessibility landscape, resulting in increased transcription of activation-specific genes (including Akt1/2,Ccnb2, and Rps6) and decreased transcription of quiescence-specific genes (including p21 and Gata2), leading to excessively activated Akt-mTOR signaling and elevated metabolism of HSC. Targeting the Akt-mTOR pathway efficiently abrogated the impaired quiescence and the increased metabolism of HSC in DEK-deficient mice, and partially rescued the long-term functions of HSC. Further, DEK regulated chromatin accessibility of HSC by recruiting the co-repressor NCoR1 to repress acetylation of histone 3 at lysine 27. Collectively, our findings revealed crucial functions of DEK in HSC quiescence maintenance and disclosed a new link between chromatin remodelers, epigenetic modification, gene transcription, and HSC homeostasis. Disclosures No relevant conflicts of interest to declare.
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Pop, Ramona, Jeffrey R. Shearstone, Qichang Shen, Ying Liu, Kelly Hallstrom, Miro Koulnis, Joost Gribnau, and Merav Socolovsky. "A Key Commitment Step In Erythropoiesis Is Synchronized with the Cell Cycle Clock through Mutual Inhibition Between PU.1 and S-Phase Progression." Blood 116, no. 21 (November 19, 2010): 839. http://dx.doi.org/10.1182/blood.v116.21.839.839.
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Abstract Abstract 839 Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are functionally coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We used flow cytometry and the cell surface markers CD71 and Ter119 to subdivide freshly isolated fetal liver cells into a developmental sequence of six subsets, from the least mature, Subset 0 (S0), to the most mature, Subset 5 (S5). We found that the upregulation of cell surface CD71, which marks the transition from S0 (CD71lowTer119negative) to S1 (CD71highTer119negative), identifies a key S-phase dependent step in erythropoiesis, that precedes, and is essential for, expression of erythroid-specific genes (Figure 1). Specifically, we found that erythroid progenitors at the transition from S0 to S1 are tightly synchronized in S-phase of the last generation of erythroid colony-forming cells (CFU-e). DNA replication within this, but not subsequent cycles, was required for a number of simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the b-globin locus control region (LCR). The S-phase dependent chromatin switch at the b-globin LCR was characterized by the formation of DNase I hypersensitivity sites, by a change in the timing of replication of the b-globin locus, by altered covalent modifications of histone tails, and by the rapid onset of DNA demethylation. An arrest of S-phase progression during the transition from S0 to S1 arrested the formation of DNase I hypersensitivity sites and prevented DNA demethylation. It also halted the subsequent transcription of b-globin and other erythroid genes. By contrast, an arrest of S-phase progression in cells that had already traversed the S0/S1 transition, no longer interfered with the erythroid transcriptional program. Mechanistically, we found that S-phase progression during this key committal step was dependent on downregulation of the cyclin-dependent kinase p57KIP2, and in turn caused the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel regulatory role for a cyclin-dependent kinase inhibitor early in erythroid maturation, distinct to their known function in cell cycle exit at the end of terminal differentiation. Furthermore, we identified a novel, mutual inhibition between PU.1 expression and S-phase progression, that provides a “synchromesh” mechanism, “locking” the erythroid differentiation program to the cell cycle clock and ensuring precise coordination of critical differentiation events. Figure 1: Regulation of the S0 to S1 transition in fetal liver erythropoiesis. Multiple differentiation milestones are synchronous with early S-phase in the last CFU-e generation (CFU-elast, black arrow), and are dependent on DNA replication. Figure 1:. Regulation of the S0 to S1 transition in fetal liver erythropoiesis. Multiple differentiation milestones are synchronous with early S-phase in the last CFU-e generation (CFU-elast, black arrow), and are dependent on DNA replication. Disclosures: No relevant conflicts of interest to declare.
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Henikoff, Steven. "New Approaches for Mapping Epigenome Dynamics." Blood 126, no. 23 (December 3, 2015): SCI—21—SCI—21. http://dx.doi.org/10.1182/blood.v126.23.sci-21.sci-21.
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Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.
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Huang, Joe Chin-Sun, Julia Sidorova, Sylvia Chien, Jin Dai, Ben Logsdon, Su-In Lee, Raymond J. Monnat, and Pamela S. Becker. "Mini-Chromosome Maintenance (MCM) DNA Helicase Genes Influence Acute Myeloid Leukemia (AML) Replication and Response to Chemotherapy-Induced DNA Damage." Blood 126, no. 23 (December 3, 2015): 3629. http://dx.doi.org/10.1182/blood.v126.23.3629.3629.
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Abstract Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic stem/progenitor cells (HSCs). For older adults (≥60 yo) with AML, the prognosis is poor. The functional decline of HSCs with aging has been linked to increased replication stress from decreased expression of mini-chromosome maintenance (MCM) DNA helicase complex proteins. As AML incidence sharply rises with age, we explored age-related differences in gene expression of MCM and RECQ DNA helicases and DNA damage response (DDR) genes in AML patient blood and bone marrow samples. We hypothesized that older AML patients would show differences in DNA replication and DDR pathways compared to younger patients. We began with an analysis of the TCGA AML database for MCM and RECQ helicase gene aberrations and found these in 37% (61/166) of the cases, with a median age of 60 years. There was reduced 5-year overall survival, 8.2 vs. 26.3 months, for those with vs. without such defects. Only a few mutations occur in these genes, with majority of aberrations due to mRNA up-regulation. 30 AML patient samples were obtained at diagnosis (n = 24) or relapse (n = 6). Total RNA was extracted from AML blasts and gene expression examined with the Affymetrix U133 Plus 2 arrays. Patients were categorized as older (≥65 yo), middle-aged (50-64 yo) and younger (<50 yo). Ingenuity Pathway Analysis (IPA) was performed on differentially expressed genes between the age-groups. FACS was used to analyze the effect of cytokine stimulation (G-CSF, IL-3, SCF) +/- 5 µM mitomycin C (induces double stranded DNA breaks) on levels of BrdU, γ-H2AX and cleaved-PARP as measures of cell cycle activity, DNA damage and apoptosis, respectively, in AML blasts. Older AML patients showed increased expression of the MCM and RECQ DNA helicases and in multiple genes involved in the DDR response, including Ataxia telangiectasia mutated (ATM)/ATM- and RAD3-related (ATR) signaling, homologous recombination (HR), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and Fanconi anemia (FA) families. Increased expression of the MCM, RECQ helicase and DDR genes in AML blast cells conferred a significantly worse prognosis. Basal γ-H2AX levels were increased in AML patients with abnormal or complex karyotype vs. normal karyotype. IPA showed age-related changes in ERG transcription factor activity, NFκB signaling and histone H3 modification. AML cells with high vs. low MCM gene expression differed in their response to growth factor stimulation and MMC treatment, in that the low MCM3 gene expressors did not progress through cell cycle after treatment with myeloid growth factors, and thus were spared of DNA damage and induction of apoptosis with MMC treatment. By Western blot analysis compared to actin control, the low MCM3 gene expressors also exhibited a trend toward significant positive correlation with MCM3 protein levels (Pearson r = 0.7, p = 0.06) In summary, older AML patients exhibited increased expression of MCM and RECQ helicases and multiple DDR genes compared to middle-aged and younger patients, and there were age-associated changes in ERG transcriptional activity, NFkB signaling and histone H3 epigenetic regulation of gene expression. These elements are thus potential targets for future drug development, particularly for older adults with AML. Disclosures No relevant conflicts of interest to declare.
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Liang, Zhuobin, Yaqun Teng, Jingchun Liu, Simonne Longerich, Xiaoyong Chen, Allison M. Green, Natalie Collins, Li Lan, Patrick Sung, and Gary M. Kupfer. "FANCI-FANCD2 Binds RNA, Which Stimulates Its Monoubiquitination." Blood 132, Supplement 1 (November 29, 2018): 645. http://dx.doi.org/10.1182/blood-2018-99-118863.
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Abstract Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and a strong cancer predisposition. FA cells are hypersensitive to DNA replicative stress, and accumulate co-transcriptional R-loops. Previous work has demonstrated that BRCA2 binds to R loops, and increased R loops are noted in FA-D2 mutant cells. Additionally, it is understood that at least one FA protein, FANCA, binds RNA. The goal of this study was to understand the relationship between FANCD2 and RNA, especially with regard to manifestation of R loops as a part of the pathophysiology of FA. First, we confirmed the increased presence of R loops in FA mutant cells using the S9.6 monoclonal antibody immunofluorescence microscopy. RNAseH overexpression removes R loop signal and increases cell survival upon mitomycin C treatment. We also showed the presence of increased R loops in an actively transcribed region of the actin gene by bisulfite DNA sequencing. We used the Damage At RNA Transcription (DART) assay, which is designed to combine oxidative DNA damage and the genomic insertion of a hyper transcription site (Fig A). Coactivation of transcription and DNA damage results in colocalization of FANCD2 and S9.6/R loop signal at the transcriptional site (Fig B and C). Consistent with the S9.6 IF, wild type RNAseH overexpression resulted in the abrogation of FANCD2 colocalization. We then asked if FANCD2 binds RNA. FANCD2 in cell lysate bound to biotinylated RNA species, preferring GC rich RNAs. Using recombinant FANCI-FANCD2 (ID2) protein (Fig D), we found that ID2 binds preferably to single stranded RNA in a more robust manner than DNA (Fig E and F). Interestingly, an ID2 complex with a known DNA binding mutation in FANCI also was defective for RNA binding. Furthermore, ID2 bound to R loops but was mediated via the single stranded DNA component of the structure. Importantly, an in vitro monoubiquitination reconstitution system using FANCL as the E3 ligase demonstrated that monoubiquitination of ID2 was stimulated to an equal or greater degree by RNA versus DNA, with greater signal in presence of GC-rich, single-stranded RNA as well as R loops (Fig G and H and data not shown). Collectively, our results support a novel mechanism the ID2 complex suppresses the formation of pathogenic R-loops by binding RNA species, thereby activating the FA pathway (Fig I). Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Walsby, Elisabeth J., Steven Coles, Steven Knapper, Chris Pepper, and Alan K. Burnett. "Topoisomerase II Inhibitor Voreloxin Causes Cell Cycle Arrest and Apoptosis in Acute Myeloid Leukaemia Cells and Acts in Synergy with Cytarabine." Blood 114, no. 22 (November 20, 2009): 4152. http://dx.doi.org/10.1182/blood.v114.22.4152.4152.
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Abstract Abstract 4152 Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. This enzyme functions as a homodimer to modulate DNA supercoiling and the unknotting and untangling of DNA. It acts via the creation of transient double strand breaks in the DNA that allow the resolution of DNA tangles prior to the rejoining of the double strand breaks. In cells with insufficient topoisomerase II activity DNA remains entangled resulting in reduced gene transcription. Conversely if a cell has excessive topoisomerase II activity the cleavage intermediates it forms with DNA can be converted into permanent strand breaks resulting in the loss of DNA integrity. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double strand breaks. These agents have been frontline drugs for the treatment of leukaemia for many years. Voreloxin (formerly SNS-595) is a first-in-class anticancer quinolone derivative that intercalates DNA and poisons topoisomerase II, inducing replication-dependent, site-selective DNA double-strand breaks. Primary acute myeloid leukaemia (AML) blasts isolated from patients at diagnosis (n = 88) had a mean LD50 (± SD) for voreloxin of 2.30μM (± 1.87). The mean Ara-C LD50 was 4.90μM (± 5.00) in the same population while the myeloid cell lines, NB4 and HL-60, had LD50 values for voreloxin of 0.23μM and 0.94μM respectively. The lower LD50 values for voreloxin in the cell lines is likely to be due to the fact that they are more actively dividing in culture than primary AML blasts and this agent is, at least to some extent, replication-dependent. Synergy experiments between voreloxin and Ara-C, (voreloxin1:2 Ara-C) identified synergism in 22 of 25 primary AML samples tested, with a mean combination index of 0.79. Apoptosis, measured by increases in Annexin V/propidium iodide (PI) staining and caspase-3 activation, was shown to increase in a dose-dependent manner. Annexin V/PI positivity was significantly increased by concentrations of voreloxin over 0.06μM (P = 0.02) while caspase-3 activation was evident at concentrations of voreloxin greater than 0.25μM (P = 0.0009). Furthermore, voreloxin was active in the p53 null K562 cell line, showing a dose-dependent increase in Annexin V/PI staining and an LD50 0.52μM. These data agree with previous reports suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. In the presence of voreloxin the ability of topoisomerase II to relax a supercoiled DNA substrate was reduced in a dose-dependent manner. Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of AML; a disease with poor long term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and AraC recommend it for further investigation Disclosures: No relevant conflicts of interest to declare.
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Tüfekçi, Özlem, Melis Kartal Yandım, Hale Ören, Gülersu Irken, and Yusuf Baran. "Targeting FOXM1 Transcription Factor In T-Cell Acute Lymphoblastic Leukemia." Blood 122, no. 21 (November 15, 2013): 4974. http://dx.doi.org/10.1182/blood.v122.21.4974.4974.
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Abstract The Forkhead box protein M1(FoxM1) is an important transcriptional factor that takes play in regulation of cell cyle, proliferation, DNA repair, apoptosis, and angiogenesis. FoxM1 overexpression has been reported to be related with many types of cancer. Since many studies have reported that FOXM1 is an important target for cancer therapy, many researchers are studying on the identification of FOXM1 inhibitors. Siomycin A, a thiazol antibiotic, is known to inhibit FoxM1 transcriptional activity. Dexamethasone is a glucocorticoid that is very important in treatment of acute lymphoblastic leukemia (ALL) and is known to be more potent compared to other steroids in the treatment of T-cell ALL. In this study, our aims were to determine the gene expression levels of FoxM1 in Jurkat cells (T-ALL cell line), to find out the possible synergistic and apoptotic effects of siomycin A and dexamethasone on this cell line and to investigate the changes in expression profiles of some important genes that have vital roles in cellular processes by targeting FoxM1 with siomycin A and dexamethasone on Jurkat cells. The gene expression levels of FoxM1 were studied with reverse transcriptase polymerase chain reaction (RT-PCR). The cytotoxic effects of siomycin A and dexamethasone on Jurkat cells were assesed by MTT cell proliferation test. The possible synergistic, additive, neutral, and antagonistic effect of combination of dexamethasone and siomycin A was determined with isobologram analysis. The apoptotic effects of these two agents were evaluated by Caspase-3 activity, loss of mitochondrial membrane potential and localisation of phosphatidilserine on plasma membrane. For this purpose, Caspase-3 calorimetric assay kit, JC-1 mitochondrial membrane potential assay kit, and Annexin V-FITC apoptosis detection kit were used, respectively. For cell cycle analysis, Jurkat cells treated with siomycin A alone or in combination with dexamethasone were stained by propidium iodide and then analyzed by flow cytometry. Expression profiles of Jurkat cells treated with siomycin A alone or in combination with dexamethasone were determined by Cancer Pathway Finder PCR Array. We found that FoxM1 gene is overexpressed in T-ALL cell line and dexamethasone and siomycin A caused a reduction in gene expression levels of FoxM1 in Jurkat cells. 8% to 13% decrease in proliferation of Jurkat cells were observed when these cells were treated with 1 and 10 µM doses of dexamethasone for 72 hours, respectively. The same doses of dexamethasone combined with siomycin A caused 74% and 75% decrease in proliferation of Jurkat cells. Isobologram analysis revealed very strong synergy between dexamethasone and siomycin A. Apoptotic tests showed no apoptotic activity of dexamethasone and siomycin A on Jurkat cells. Cell cycle analysis demonstrated that, reduction of FOXM1 expression by combination of dexamethasone and siomycin A in Jurkat cells inhibited cell proliferation through induction of G1 phase arrest. PCR Array results showed that apoptotic CASPASE-2, CASPASE-7, and CASPASE-9 genes and XIAP and CYCLIN D3 genes were upregulated in response to the treatment. ETS2 gene, which is known as a protooncogene and shown to be involved in regulation of telomerase, was downregulated in response to siomycin A and dexamethosone alone and in combined treatment. TERF1 gene, which inhibits telomerase activity, was upregulated by the treatment. Combination of Siomycin A and dexamethasone downregulated the MCM-2 gene, which is a key component of the pre-replication complex and involved in the formation of DNA replication fork. Moreover, combined treatment resulted in the downregulation of MKI67 gene encoding a nuclear protein associated with cellular proliferation. WEE1 gene, which inhibits G2/M phase transition in cell cycle, was also upregulated. These data indicate that FoxM1 gene is strongly overexpressed in T-ALL cell line and targeting FoxM1 by siomycin A and dexamethasone causes a significant decrease in T-ALL cell proliferation through induction of G1 cell cycle arrest. Importantly, PCR array analyses also showed that siomycin A and dexamethasone treatment affects Jurkat cells via upregulating or downregulating the key genes of cell cycle, apoptosis, cell proliferation, telomere, and telomerase function. All these findings suggest a possible role for FoxM1 in T-ALL pathogenesis and represent FoxM1 as an attractive target for T-ALL therapy. Disclosures: No relevant conflicts of interest to declare.
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Pippa, Raffaella, Ana Dominguez, Nerea Marcotegui, Raquel Malumbres, Elizabeth Guruceaga, and Maria D. Odero. "RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 879. http://dx.doi.org/10.1182/blood.v124.21.879.879.
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Abstract INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation. AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation. RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene. CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias. Disclosures No relevant conflicts of interest to declare.
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Chae, Hee-Don, Bryan Mitton, and Kathleen Sakamoto. "CREB Regulates Cell Cycle Progression through RFC3-PCNA Axis in Acute Myeloid Leukemia." Blood 124, no. 21 (December 6, 2014): 881. http://dx.doi.org/10.1182/blood.v124.21.881.881.
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Abstract CREB (cAMP Response Element Binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication Factor C3 (RFC3), a 38 kDa subunit of the RFC complex, is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in DNA replication and repair processes. CREB1 expression was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line, suggesting that RFC3 and CREB1 might be target genes of E2F, a key transcriptional regulator of the G1/S progression. Though there were two potential E2F binding sites in the RFC3 promoter region, chromatin immunoprecipitation assays provided no evidence for E2F1 binding to the RFC3 promoter, whereas E2F1 could directly act on the CREB1 expression. Treatment with the cyclin-dependent kinase (CDK) inhibitor AT7519 decreased expression of CREB1 and RFC3 as well as well-known E2F target genes such as CCNE1, CCNA2 and CCNB1 in KG-1 cells. These results indicate that CREB1 overexpression, a potentially important prognostic marker in leukemia patients, may be associated with dysregulated CDK-E2F activity in leukemia. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in AML cells from patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB knockdown primarily inhibited G1/S cell cycle transition, decreasing expression of RFC3 as well as PCNA loading onto chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading [Chromatin-bound PCNA-positive cells (%), control vs. CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 8h after release from mitotic arrest: 66.87 +/– 0.90 vs. 24.77 +/– 0.99 vs. 79.17 +/– 0.12, n=3, p< 0.01, mean +/– SEM] caused by CREB knockdown. Taken together, our results suggest that RFC3 may play a role in neoplastic myelopoiesis by promoting the G1/S progression, and its expression is regulated by CREB. These data provide new insight into CREB-driven regulation of the cell cycle in AML cells, and may contribute to leukemogenesis associated with CREB overexpression. Disclosures No relevant conflicts of interest to declare.
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Wu, Xue, Yuan Li, and Baoan Chen. "Integrated Analysis of Key Genes for FGFR1 Knockdown in Mantle Cell Lymphoma Cell Line (Z-138)." Blood 136, Supplement 1 (November 5, 2020): 32. http://dx.doi.org/10.1182/blood-2020-143311.
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Background:Mantle cell lymphoma (MCL) is the secondary common B cell lymphoma subtype that comprises 6 to 8% of non-Hodgkin's lymphoma, and is closely related to the poor clinical outcomes. Previous studies have focussed on the mechanisms mediating ibrutinib resistance, the first-in-class oral covalent inhibitor of Bruton's tyrosine kinase (BTK). The aims of the this study is to identify key genes related to the FGFR1 Knockdown in Mantle cell lymphoma cell line (Z-138) that has been proved to play a vital role in MCL progression. Methods:GSE138127 mRNA microarray datasets from Gene Expression Omnibus (GEO) were analysed to obtain differentially expressed genes (DEGs) between vector control and the Knockdown of FGFR1 charactered by ibrutinib resistance. The GO and GSEA were carried out by WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) to do the functional enrichment analysis. The network analysis of protein-protein interactions (PPIs), TF-gene interaction were carried out by Network Analyst 3.0 to identify hub genes. And the main hub gene was probed using Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Results:In total, 175 DEGs were obtained, of which 87 and 88 were up- and down-regulated, respectively. Three hub genes (CDK1, CCND1) were identified and associated to cell cycle and DNA replication. FOXC1 were identified as the potential Transcription factors in the biological process. Conclusion:CDK1, CCND1 may affect the cell cycle regulated by FOXC1, and represent the new candidate molecular markers of the occurrence of ibrutinib resistance. Keywords: Mantle cell lymphoma (MCL), ibrutinib resistance, Network Analyst, Microarray, Protein-protein interactions, Molecular markers Disclosures No relevant conflicts of interest to declare.
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Lingeman, Robert G., Robert J. Hickey, and Linda H. Malkas. "Abstract 1875: Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor." Cancer Research 84, no. 6_Supplement (March 22, 2024): 1875. http://dx.doi.org/10.1158/1538-7445.am2024-1875.
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Abstract AOH1996 targets a cancer-associated form of PCNA (caPCNA) by altering the protein-protein interface between caPCNA and its many binding partners. AOH1996 does this by inserting into the PCNA-interacting protein-box (PIP-box) pocket that is, in part, defined by the interdomain connecting loop (IDCL), the site of interaction between PCNA and most of its many binding partners. As a result, AOH1996 inhibits PCNA functions such as DNA replication, DNA repair, and transcription-replication conflict (TRC) resolution leading to cell cycle arrest and apoptosis. These effects are cancer specific and AOH1996 has little effect on non-cancerous cells even at 6-fold the effective dose in cancer cells. AOH1996 was developed in our lab and is currently in Phase I human clinical trials. Six patients have been enrolled in the trial and have not experienced AOH1996-related toxicities as the doses have been escalated to levels found to be efficacious in preclinical animal studies. Resistance to EGFR tyrosine kinase inhibitors (TKIs) poses a significant challenge in the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Initially, EGFR TKIs like gefitinib, erlotinib, afatinib and osimertinib deliver remarkable responses, inducing tumor regression and improving overall survival. However, the emergence of resistance mechanisms, such as secondary EGFR mutations and bypass signaling pathway activation, hampers the efficacy of these drugs over time. The functional relationship between PCNA and EGFR has implications for the treatment of lung cancer patients with activating EGFR mutations. Membrane-localized EGFR modulates the PCNA-centric process of DNA replication through signaling pathways such as the Ras-Raf-Mek-Erk, PI3K/AKT, and PLCγ1/PKC signaling pathways. Nucleus-localized EGFR enhances PCNA stability by phosphorylating PCNA on tyrosine 211 (Y211), which prevents its degradation. In addition, phosphorylation at Y211 by EGFR results in decreased association of PCNA with MutS, which results in reduced mismatch repair (MMR). In this study, we combine AOH1996 and osimertinib as an innovative approach to treating NSCLC cancers with activating EGFR mutations and find that the drug combination: 1.) enhances the killing of NSCLC cell lines with activating EGFR mutations and acquired resistance to TKIs; 2.) enhances the destabilization of PCNA on chromatin; and 3.) causes changes in the localization and colocalization of PCNA and EGFR. We conclude that the combination of AOH1996 and osimertinib holds much potential as an improved treatment regimen for lung cancer patients with activating EGFR mutations. Citation Format: Robert G. Lingeman, Robert J. Hickey, Linda H. Malkas. Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1875.
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Timofeev, Nadia, Jacqueline N. Milton, Stephen W. Hartley, Richard Sherva, Paola Sebastiani, Daniel A. Dworkis, Elizabeth S. Klings, et al. "Genome-Wide Studies in Sickle Cell Anemia Show Associations Between SNPs in the Olfactory Receptor Gene Cluster and Fetal Hemoglobin Concentration." Blood 114, no. 22 (November 20, 2009): 821. http://dx.doi.org/10.1182/blood.v114.22.821.821.
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Abstract Abstract 821 Fetal hemoglobin (HbF) is the major modulator of sickle cell anemia (SCA, homozygosity for HBB glu6val) severity. In a genome-wide association study in African Americans with SCA we sought to identify single nucleotide polymorphisms (SNPs) associated with HbF concentrations. A discovery sample of 848 African American subjects and a primary replication study of 305 subjects were examined. DNA was genotyped with the Illumina Human610-Quad SNP; some replication set samples were genotyped with the Sentrix HumanCNV370 or the 317K array. For quality control we excluded SNPs with a call rate less than 95%; we excluded subjects with a call rate less than 93%; identity by descent measurements were computed to identify related individuals who were removed from analysis; we inferred gender using chromosome X SNPs removing subjects with gender mismatches; a genome-wide principal components analysis found no association between the phenotype and the first 10 principal components, indicating that the results were not affected by population substructure. The association between HbF and the genotype for each SNP was tested with a multiple linear regression analysis adjusting for sex and assuming an additive model using the software PLINK. SCA is a rare disease in developed countries and assembling large data sets is not feasible. Therefore, true associations with limited effect sizes might not reach “genome-wide” significance of 10-08. To identify genes enriched with moderately strong associations, we developed a SNP set enrichment analysis (SSEA) that computes the probability that a set of SNPs is selected as significant by chance and scores each gene by this probability. Two SNPs exceeded the strict genome-wide significance: SNP rs5006884 in a novel region on chromosome 11 upstream of the β-globin gene cluster locus control region (LCR) containing the olfactory receptor (OR) genes OR51B5 and OR51B6; SNP rs766432 in BCL11A, previously found to be associated with HbF in several different populations. Data for SNPs common to the discovery and replication sets were combined and analyzed jointly. Similarity of the regression beta coefficients across datasets and increased significance of the p-values compared with those observed in the analyses of individual datasets provide additional evidence that the associations were consistent in the both datasets. The Q-Q plot and a genomic inflation factor of 1.003 both suggest that the test statistics are not inflated and are distributed appropriately. SSEA identified 2 OR genes (OR51B5, OR51B6) and BCL11A as enriched in both the discovery and replication sets. The most significant SNP in the OR region (rs5006884) and BCL11A (rs766432) explained 15.6% of the variability in HbF. Also, in the interval Xp 22.2-22.3 we found moderate, but not “genome-wide” significance for 1 SNP in Xp22.2. Phylogenetic conservation of some OR genes and their flanking sequences suggests that this region might also have a role in controlling expression within the β-globin gene-like complex. Low linkage disequilibrium between SNPs in the β-globin locus and the OR genes suggests that one or more variants in the OR genes independently regulate HbF. The top SNP in the OR51B5/OR51B6 locus, rs5006884, was still associated with HbF (p = 1.5E-05) in a model adjusting both for sex and rs2071348, a SNP in tight LD with the HBG2 5' -158 C-T SNP, giving further evidence that the OR region provides important information in addition to the SNPs in the β-globin gene-like complex. Polymorphisms in the upstream OR region might conceivably modulate HbF levels by altering chromatin structure within the β-globin gene cluster. Conserved binding sites for the transcription factor CTCF flank the β-globin gene cluster and evidence suggests that these sites function as insulators. Polymorphisms in this region might affect the actions of enhancers, possibly through their effects on CTCF binding its receptors, thereby affecting the interaction of the globin genes with enhancers in the OR regions. Disclosures: No relevant conflicts of interest to declare.
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Viziteu, Elena, Yea Lih Lin, Laure Vincent, Anja Seckinger, Dirk Hose, Angelos Constantinou, Bernard Klein, Philippe Pasero, and Jerome Moreaux. "A Small Molecule That Selectively Targets BLM Helicase Has a Therapeutic Interest in Multiple Myeloma." Blood 128, no. 22 (December 2, 2016): 4433. http://dx.doi.org/10.1182/blood.v128.22.4433.4433.
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Abstract Hematological malignancies, including MM, are characterized by genomic instability that is possibly related to underlying defects in DNA repair or genomic stability maintenance. RECQ helicases are at the crossroad between replication, recombination, DNA repair and transcription and could represent potent therapeutic targets for cancer therapy. The Bloom's syndrome helicase, BLM, has DNA annealing and unwinding activities. Through its interaction with TOPOIIIα, BLM unwinds the short stretches of naked duplex DNA and processes homologous recombination (HR) intermediates containing a double holiday junction. It may suppress hyper-sister chromatid exchange (SCE) by disruption of D-loop recombination intermediates and also might be involved in the suppression of crossing over during homology mediated recombination. This helicase facilitates telomere replication by resolving G4 structures. BLM deregulation is also associated with cancer. We recently identified a significant overexpression of BLM in malignant plasma cells compared to normal bone marrow plasma cells. Since BLM, plays important roles in the maintenance of chromosomal stability, we investigated if BLM could play a role in MM pathophysiology and drug resistance. A high BLM expression in MMCs could predict for shorter overall survival (OS) in four independent cohorts of patients (P = 0.003 in the HM cohort (N=206), P = 0.0002 in the UAMS-TT2 cohort (N=345), P = 0.0008 in TT3 cohort (N=186)) and P = 0.04 in Hovon cohort (N=282). A high BLM expression in MMCs could also predict for shorter event free (EFS) survival in the HM and UAMS-TT2 cohorts. BLM expression was significantly higher in the poor prognosis proliferation MM subgroup (P < 0.05). Moreover, we found that BLM was overexpressed in HMCLs (median 1848, range 246 - 10901) compared to primary MMCs or BMPCs (P< .001). The small molecule, ML216, is a membrane permeable selective inhibitor of BLM helicase. ML216 appears to act at the BLM-nucleic acid substrate binding site, inhibiting DNA binding and blocking BLM's helicase activity. We investigated the interest of the BLM inhibitor, ML216, to eradicate MM cells. The effect of ML216 was tested using 10 different human myeloma cell lines (HMCL). ML216 induced a dose dependent inhibition of cell growth in all investigated HMCL with a median IC50 of 4.1 μM (range: 1.2 - 30 μM). Moreover, our tests on HMCL showed that the BLM inhibitor synergizes with Melphalan and Bortezomib, two drugs currently used in myeloma therapy. Furthermore, ML216 induced a significant apoptosis of primary myeloma cells of patients co-cultured with their bone marrow environment and recombinant IL-6 (n=7). BLM inhibitor significantly reduced the median number of viable myeloma cells by 54%, 60% and 74% at respectively 3, 6 and 10 μM (P = 0.001, P < 0.001 and P<0.001 respectively; n=7). Of interest, the normal non myeloma cells present in the culture were significantly less affected by ML216 when used at 3 and 6 μM. These data demonstrated a significant higher toxicity of ML216 on myeloma cells compared to normal bone marrow cells. Taken together, our results underline the therapeutic interest of BLM inhibitor in MM and especially in patients characterized by high BLM expression and a poor prognosis. Disclosures No relevant conflicts of interest to declare.
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Wells, James P., Emily Yun-Chia Chang, Leticia Dinatto, Justin White, Stephanie Ryall, and Peter C. Stirling. "RAD18 opposes transcription-associated genome instability through FANCD2 recruitment." PLOS Genetics 18, no. 12 (December 8, 2022): e1010309. http://dx.doi.org/10.1371/journal.pgen.1010309.
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DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts.
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Boddu, Prajwal, Abhishek Gupta, Rahul Roy, Anne Olazabal Herrero, Amit Verma, Karla Neugebauer, and Manoj Pillai. "Transcription Defects in SF3B1K700E Induce Targetable Alterations in the Chromatin Landscape." Blood 142, Supplement 1 (November 28, 2023): 709. http://dx.doi.org/10.1182/blood-2023-188083.
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Introduction: Acquired mutations, most commonly in RNA splicing factors and epigenetic regulators are thought to be disease drivers of clonal myeloid disorders such as MDS and AML. Both MDS and AML are amenable to epigenetic therapies regardless of underlying mutational subtypes. This prompted our hypothesis that alterations to epigenetic and chromatin landscape is important in pathobiology of clonal myeloid disorders driven by splicing factor mutations. In this study, we demonstrate that SF3B1 mutations induce distinct changes to epigenetic landscape and chromatin organization by altering RNA Polymerase II (Pol2) transcription kinetics. Critically, such epigenetic changes are also targetable - inhibiting components in the H3K4me/Sin3/HDAC pathway reverses Pol2 transcription defects and downstream effects on chromatin organization and genomic integrity. Methods: We generated inducible isogenic K562 cell lines that express single mutant allele of SF3B1 K700Ecombining two genome editing approaches (AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021)) (Fig1A). RNAPII transcription kinetics changes were assessed using ChIP-seq for Pol2 and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing (TT-TL-seq). Chromatin accessibility/nucleosome density and histone marks (H3K4me3, H3K27ac, H3K27me3) were assessed using ATAC-seq and low-input CUT&RUN, respectively. Key results were replicated in patient samples with SF3B1 mutations and in a Sf3b1K700E mouse model. Results: Combining genome-wide approaches (ChIP-seq (Fig1B), GRO-seq, and TT-TL seq (Fig1C)), we found that Pol2 in SF3B1 K700E redistributes into the gene body due to a primary gene-body elongation defect. This transcriptional dysregulation was reflected in downstream effects such as excess R-loops (S9.6 microscopy; Fig1D), transcription-replication conflicts (proximity ligation assay for PCNA-Pol2; Fig1E), and S-phase arrest (flow cytometry; Fig1F). Importantly, normalized Pol2 density at proximal-promoter (p-p) region was reduced in SF3B1 K700E (Fig1G). This correlated with a reduction in p-p H3K4me3 in SF3B1 K700E determined by Cut and Run (Fig1H). ATAC-seq showed a reduction in promoter accessibility (Fig1I) and increased nucleosome promoter density (Fig1J). ChIP-seq for CDK9, a component of p-TEFb pause release complex, showed higher occupancy at promoters in SF3B1 K700E (Fig1K), suggesting that premature promoter pause release of Pol2 is the major driver for the observed reduction in p-p Pol2 density. Consistent with the importance of p-p Pol2 in maintaining accessible chromatin states, our findings suggest that depletion of p-p Pol2 leads to nucleosome repositioning at promoters and loss of chromatin accessibility (Gilchrist et al, Cell, 2010). Given the extensive cross talk between chromatin and transcription, we speculated that modulating epigenetic regulators might improve Pol2 elongation defects and rescue cells from downstream effects. An unbiased shRNA rescue screen, targeting epigenetic regulators and chromatin modifier protein classes, enriched for proteins in the Sin3/HDAC pathway (ING2, HDAC2) (Fig1L). Conversely, knocking down (KD) for WDR5 (H3K4me3 writer complex) further worsened survival. HDAC2 and ING2 KD cells showed reduced R-loops and DNA damage, lower gene-body Pol2 ChIP densities (Fig1M), normalization of p-p H3K4me3 densities (Fig1N), and rescue from S-phase arrest. These effects of WDR5 and HDAC2/ING2 KD on survival in SF3B1-mutant isogenic cells was confirmed in both MDS patient samples (Fig1O)- and Sf3b1K700E murine models (Fig1P)- contexts, using in vitro colony assays. Conclusion: Our findings show that SF3B1 K700E-induced disruption of Pol2 elongation kinetics is reflected in reduced Pol2 density at proximal promoter sites and results in a corresponding reduction in chromatin accessibility and promoter H3K4me3. Through an unbiased shRNA screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, improved the transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes affect chromatin organization through their effects on Pol II transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.
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St Germain, Commodore, Hongchang Zhao, and Jacqueline H. Barlow. "Transcription-Replication Collisions—A Series of Unfortunate Events." Biomolecules 11, no. 8 (August 21, 2021): 1249. http://dx.doi.org/10.3390/biom11081249.
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Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.
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Levy, Emily R., Joseph Clara, David Allan, Robert Reger, and Richard W. Childs. "CRISPR Gene-Editing of Chemokine Receptors As a Novel Strategy to Redirect NK Cell Trafficking In Vivo." Blood 136, Supplement 1 (November 5, 2020): 3. http://dx.doi.org/10.1182/blood-2020-141408.
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Allogeneic and autologous natural killer (NK) cell-based therapies have proven to be safe in patients with hematological malignancies across multiple clinical trials. However, at present, only a small percentage of patients who have received NK cell based therapy have achieved complete remissions. NK cells must first traffic into the tumor microenvironment in order to effectively lyse tumor targets in vivo. Many hematological malignancies reside in or metastasize through bone marrow (BM) compartments. Recent studies in both murine and macaque models evaluating NK cell distribution following intravenous (i.v.) infusion unexpectedly revealed preferential trafficking into liver tissue over BM niches, which for hematological malignancies is undesirable. Therefore, we evaluated surface expression and mRNA transcript abundance of critical chemotactic receptors and adhesion molecules in primary fresh and ex vivo expanded NK cells. NK cells from six healthy donor PBMC samples were expanded for 14 days ex vivo, using two different irradiated feeder cell lines (either EBV-infected LCL or K562 cells with membrane-bound IL-21 and 41BBL). mRNA from fresh and expanded NK cells was sequenced and analyzed for differential gene expression. We observed the transcriptome of NK cells expanded with the two different feeder cell lines were similar, with only 13 differentially expressed genes. However, we observed the transcriptional landscape between fresh and expanded NK cells to be vastly different (Figure 1A). As expected, amongst the thousands of differentially expressed genes, mitotic phase transitions and DNA replication were pathways that were strongly enriched in expanded compared to fresh NK cells. Further, expanded NK cells had a robust amplification in mRNA transcription of known surface cytotoxicity and activation receptors, as well as enhanced mRNA transcription of chemotactic ligands that are implicated in bridging the innate and adaptive immune responsesincluding XCL1, XCL2, CCL5, CXCL16, and CXCL8. Remarkably, we found expanded NK cells had upregulated mRNA transcription and surface expression of CCR1, CCR5, CXCR3, and CXCR6, coupled with a substantial decrease in mRNA transcription and surface expression of CXCR4 (Figure 1B). We postulate that this particular shift in molecular expression may have the net effect of routing adoptively transferred NK cells out of the circulation into liver and other inflammatory environments, ultimately impeding cellular trafficking into the bone marrow (BM). To test this hypothesis, we used CRISPR/Cas9 to disrupt the CCR5 gene which has upregulated expression in expanded NK cells. Five days following ex-vivo expansion, NK cells were electroporated with a mix of 3 sgRNAs targeting CCR5, complexed with Cas9. Electroporated cultures harvested on day 14 maintained their proliferative capacity and had a substantial reduction in CCR5 expression (22%) compared to non-electroporated control NK cells (88%; Figure 1C). Both expanded NK cell populations were then injected i.v. into NSG mice, with blood, BM, lungs, and livers being harvested 24 hours following infusion. Disrupting CCR5 in NK cells significantly reduced cellular trafficking into the liver compared to control NK cells (p<.01). Moreover, the percentage of infused CCR5 CRISPR/Cas9 disrupted NK cells in the circulation was increased compared to control NK cells (Figure 1D). In conclusion, these collective data reveal NK cells undergo profound transcriptional changes when cultivated ex vivo. Many chemotactic receptors that were previously unknown to be impacted by ex vivo expansion were discovered to have a shift in transcriptional regulation which would be predicted to compromise NK cell homing to the bone-marrow. Importantly, we show for the first time that CRISPR gene-editing of chemokine receptors can be used as a novel strategy to redirect NK cell trafficking in vivo, which could bolster the effectiveness of adoptive NK cell immunotherapy for hematological malignancies and other cancers. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Tsirkas, Ioannis, Daniel Dovrat, Manikandan Thangaraj, Ineke Brouwer, Amit Cohen, Zohar Paleiov, Michael M. Meijler, Tineke Lenstra, and Amir Aharoni. "Transcription-replication coordination revealed in single live cells." Nucleic Acids Research 50, no. 4 (February 8, 2022): 2143–56. http://dx.doi.org/10.1093/nar/gkac069.
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Abstract The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.
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Lalonde, Maxime, Manuel Trauner, Marcel Werner, and Stephan Hamperl. "Consequences and Resolution of Transcription–Replication Conflicts." Life 11, no. 7 (June 30, 2021): 637. http://dx.doi.org/10.3390/life11070637.
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Transcription–replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.
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Lovett, Susan T. "DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts." Microbial Cell 8, no. 6 (June 7, 2021): 143–45. http://dx.doi.org/10.15698/mic2021.06.753.
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In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study we isolate spontaneous suppressor mutants in a holC∆ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β’) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.
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Kogoma, T. "Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription." Microbiology and Molecular Biology Reviews 61, no. 2 (June 1997): 212–38. http://dx.doi.org/10.1128/mmbr.61.2.212-238.1997.
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Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.
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Kogoma, T. "Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription." Microbiology and molecular biology reviews : MMBR 61, no. 2 (1997): 212–38. http://dx.doi.org/10.1128/.61.2.212-238.1997.
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Bayona-Feliu, Aleix, and Andrés Aguilera. "The role of chromatin at transcription-replication conflicts as a genome safeguard." Biochemical Society Transactions 49, no. 6 (November 25, 2021): 2727–36. http://dx.doi.org/10.1042/bst20210691.
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DNA replication ensures the correct copying of the genome and the faithful transfer of the genetic information to the offspring. However, obstacles to replication fork (RF) progression cause RF stalling and compromise efficient genome duplication. Since replication uses the same DNA template as transcription, both transcription and replication must be coordinated to prevent Transcription-Replication Conflicts (TRCs) that could stall RF progression. Several factors contribute to limit the occurrence of such conflicts and their harmful impact on genome integrity. Increasing evidence indicates that chromatin homeostasis plays a key role in the cellular response to TRCs as well as in the preservation of genome integrity. Indeed, chromatin regulating enzymes are frequently mutated in cancer cells, a common characteristic of which is genome instability. Therefore, understanding the role of chromatin in TRC occurrence and resolution may help identify the molecular mechanism by which chromatin protects genome integrity, and the causes and physiological relevance of the high mutation rates of chromatin regulating factors in cancer. Here we review the current knowledge in the field, as well as the perspectives and future applications.
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Tsai, Shuhe, Louis-Alexandre Fournier, Emily Yun-chia Chang, James P. Wells, Sean W. Minaker, Yi Dan Zhu, Alan Ying-Hsu Wang, Yemin Wang, David G. Huntsman, and Peter C. Stirling. "ARID1A regulates R-loop associated DNA replication stress." PLOS Genetics 17, no. 4 (April 7, 2021): e1009238. http://dx.doi.org/10.1371/journal.pgen.1009238.
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ARID1A is a core DNA-binding subunit of the BAF chromatin remodeling complex, and is lost in up to 7% of all cancers. The frequency of ARID1A loss increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeling complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers.
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Dimude, Juachi, Monja Stein, Ewa Andrzejewska, Mohammad Khalifa, Alexandra Gajdosova, Renata Retkute, Ole Skovgaard, and Christian Rudolph. "Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome." Genes 9, no. 8 (July 27, 2018): 376. http://dx.doi.org/10.3390/genes9080376.
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The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication–transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY, integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replication–transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.
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Wu, Wei, Ian D. Hickson, and Ying Liu. "The prevention and resolution of DNA replication–transcription conflicts in eukaryotic cells." Genome Instability & Disease 1, no. 3 (May 2020): 114–28. http://dx.doi.org/10.1007/s42764-020-00012-z.
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