Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a ...role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.
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•A role for SETD2 in DSB resection and homologous recombination repair•Histone H3K36me3 is required for homologous recombination•SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining•Mutations affecting H3K36me3 levels may promote tumorigenesis
The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end ...joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from ...intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5—the exon junction positioned at U5Loop1 C
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; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial
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LtrB intron with new loci in retrotransposition—guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson–Crick pairs with the 5′exon in the absence of +5G at the intron start. In 5′exon positions −3 and −5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson–Crick pairs with 3′exon position +1 coincide with substitutions of the conserved −3C at the intron 3′end. Based on mutation and X-ray evidence, we propose that −3C pairs with U2 G
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juxtaposing the branchpoint and the 3′intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3′intron end ensure that the 3′exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson–Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson–Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.
SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, ...SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.
As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate ...cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.
Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its ...phosphorylation in an ATM‐dependent manner after DNA damage. In vitro kinase assay and site‐directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia‐derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM‐dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low‐dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment‐associated secondary leukemia following exposure to TOP2 inhibitors.
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research ...into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.
Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, ...particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iβ complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.
Topoisomerase (topo) IIα and IIβ maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as ...previously reported, but also by topo IIβ. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIβ. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIβ, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIβ inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIβ function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIβ cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.
Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely ...understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors.
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