David Llères

SUMMARY: The cell-cycle regulated methyltransferase SET8 is the sole enzyme responsible for the mono-methylation of histone H4 at lysine 20 (H4K20) that is the substrate for di- and tri-methylation mainly by SUV4-20Hs enzymes. Both SET8 and SUV4-20Hs have been implicated in regulating DNA repair pathway choice through the inverse affinities of BRCA1-BARD1 and 53BP1 complexes for disparate methylation states of H4K20. However, the precise and respective functions of each H4K20 methyltransferases in DNA repair pathways remained to be clarified. Here, we show that SET8 acts as a potent chromatin inhibitor of homologous recombination and that its timely degradation during DNA replication is essential for the spontaneous nuclear focal accumulation of BRCA1 and RAD51 complexes during S-phase. Strikingly, the anti-recombinogenic function of SET8 is independent of SUV4-20H activity but requires the subsequent recruitment of the ubiquitin ligase RNF168. Moreover, we show that SET8-induced BRCA1 inhibition is not necessarily related to the loss of BARD1 binding to unmethylated histone H4K20. Instead, it is largely caused by the accumulation of 53BP1 in a manner depending on the concerted activities of SET8 and RNF168 on chromatin. Conversely, the lack of SET8 and H4K20 mono-methylation on newly assembly chromatin after DNA replication led to the untimely accumulation of BRCA1 on chromatin at the subsequent G1 phase. Altogether, these results establish the de novo activity of SET8 on chromatin as a primordial epigenetic lock of BRCA1-mediated HR pathway during the cell cycle.

Metazoan promoters are enriched in secondary DNA structure-forming motifs, such as G-quadruplexes (G4s). Here we describe ‘G4access’, an approach to isolate and sequence G4s associated with open chromatin via nuclease digestion. G4access is antibody- and crosslinking-independent and enriches for computationally predicted G4s (pG4s), most of which are confirmed in vitro. Using G4access in human and mouse cells, we identify cell-type-specific G4 enrichment correlated with nucleosome exclusion and promoter transcription. G4access allows measurement of variations in G4 repertoire usage following G4 ligand treatment, HDAC and G4 helicases inhibitors. Applying G4access to cells from reciprocal hybrid mouse crosses suggests a role for G4s in the control of active imprinting regions. Consistently, we also observed that G4access peaks are unmethylated, while methylation at pG4s correlates with nucleosome repositioning on DNA. Overall, our study provides a new tool for studying G4s in cellular dynamics and highlights their association with open chromatin, transcription and their antagonism to DNA methylation.

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher‐order chromatin structure have remained elusive. Here, we applied FLIM‐FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called “nanocompaction.” Both in naive embryonic stem cells (ESCs) and in ESC‐derived epiblast‐like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1β, indicating the existence of a dynamic HP1‐dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular‐proliferation marker Ki‐67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki‐67.

PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1β (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1β-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1β on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization. Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on C. elegans germ cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, the C. elegans homologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, our in vivo findings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.

PA28γ, a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, similarly to that observed upon the knockdown of HP1β, a key factor in heterochromatin structure. We show that PA28γ is present at HP1β-containing repetitive-DNA sequences abundant in heterochromatin and importantly, that HP1β on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are both involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the higher order structuration of chromatin.

SUMMARY: Mammalian genomic imprinting is essential for development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. Here, we compared chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints—the Igf2-H19 and the Dlk1-Dio3 domains—and assessed the involvement of the insulator protein CTCF. At both domains, CTCF binds the maternal allele of a differentially-methylated region (DMR), in addition to multiple instances of bi-allelic CTCF binding in their surrounding TAD (Topologically Associating Domain). On the paternal chromosome, bi-allelic CTCF binding alone is sufficient to structure a first level of sub-TAD organization. Maternal-specific CTCF binding at the DMRs adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal sub-TAD organisation. Genome-editing experiments at the Dlk1-Dio3 locus confirm that the maternal sub-TADs are essential during development to maintain the imprinted Dlk1 gene in an inactive state on the maternal chromosome.

Significance: Chronic neuroinflammation and oxidative stress are likely complicit in driving disease progression in Huntington's disease (HD). Here, we describe the mechanism of action of a unique chemical scaffold that is highly selective for activation of NRF2, the master transcriptional regulator of cellular antiinflammatory and antioxidant defense genes. The use of this scaffold revealed that NRF2 activation responses were muted in HD patient-derived neural stem cells, suggesting increased susceptibility of this critical renewable cell population to oxidative stress in HD brain. However, pharmacological activation of NRF2 was able to repress inflammatory responses in mouse microglia and astrocytes, the principal cellular mediators of neuroinflammation, and in blood monocytes from HD patients. Our results suggest multiple protective benefits of NRF2 activation for HD patients.