Guillaume Guilbaud

Determinants of G quadruplex‐induced epigenetic instability in REV1‐deficient cells

REV1‐deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)‐forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4‐induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU‐1 locus in REV1‐deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus.

PrimPol Is Required for Replicative Tolerance of G Quadruplexes in Vertebrate Cells

Summary G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.

Histone H3.3 Is Required to Maintain Replication Fork Progression after UV Damage

Summary Unlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle [1] and is incorporated into chromatin independent of replication [2]. Recently, H3.3 has been implicated in the cellular response to ultraviolet (UV) light [3]. Here, we show that chicken DT40 cells completely lacking H3.3 are hypersensitive to UV light, a defect that epistasis analysis suggests may result from less-effective nucleotide excision repair. Unexpectedly, H3.3-deficient cells also exhibit a substantial defect in maintaining replication fork progression on UV-damaged DNA, which is independent of nucleotide excision repair, demonstrating a clear requirement for H3.3 during S phase. Both the UV hypersensitivity and replication fork slowing are reversed by expression of H3.3 and require the specific residues in the α2 helix that are responsible for H3.3 binding its dedicated chaperones. However, expression of an H3.3 mutant in which serine 31 is replaced with alanine, the equivalent residue in H3.2, restores normal fork progression but not UV resistance, suggesting that H3.3[S31A] may be incorporated at UV-damaged forks but is unable to help cells tolerate UV lesions. Similar behavior was observed with expression of H3.3 carrying mutations at K27 and G34, which have been reported in pediatric brain cancers. We speculate that incorporation of H3.3 during replication may mark sites of lesion bypass and, possibly through an as-yet-unidentified function of the N-terminal tail, facilitate subsequent processing of the damage.

Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

Summary Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU) can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.

Local epigenetic reprogramming induced by G-quadruplex ligands

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

Contribution of epigenetic landscapes and transcription factors to X-chromosome reactivation in the inner cell mass.

X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks.X-chromosome inactivation is reversed in the mouse inner cell mass (ICM) through a mechanism that is not fully understood. Here, the authors investigate this process and characterize the contributions of the epigenetic landscape and transcription factors in X-linked gene reactivation dynamics.

The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability

Summary Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs) depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.

Unwinding to Recombine

The MCM proteins are best known for their role in DNA replication, MCM2-7 forming the replicative helicase. Now, two reports in this issue of Molecular Cell, Nishimura et al. (2012) and Lutzmann et al. (2012) show the less well understood MCM8 and MCM9 to be crucial for effective homologous recombination.