PR-Set7–dependent lysine methylation ensures genome replication and stability through S phase

PR-Set7/SET8 is a histone H4–lysine 20 methyltransferase required for normal cell proliferation. However, the exact functions of this enzyme remain to be determined. In this study, we show that human PR-Set7 functions during S phase to regulate cellular proliferation. PR-Set7 associates with replication foci and maintains the bulk of H4-K20 mono- and trimethylation. Consistent with a function in chromosome dynamics during S phase, inhibition of PR-Set7 methyltransferase activity by small hairpin RNA causes a replicative stress characterized by alterations in replication fork velocity and origin firing. This stress is accompanied by massive induction of DNA strand breaks followed by a robust DNA damage response. The DNA damage response includes the activation of ataxia telangiectasia mutated and ataxia telangiectasia related kinase–mediated pathways, which, in turn, leads to p53-mediated growth arrest to avoid aberrant chromosome behavior after improper DNA replication. Collectively, these data indicate that PR-Set7–dependent lysine methylation during S phase is an essential posttranslational mechanism that ensures genome replication and stability

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells

Cbx2 targets PRC1 to constitutive heterochromatin in mouse zygotes in a parent-of-origin-dependent manner

Polycomb repressive complexes PRC1 and PRC2 regulate expression of genes involved in proliferation and development. In mouse early embryos, however, canonical PRC1 localizes to paternal pericentric heterochromatin (pat-PCH), where it represses transcription of major satellite repeats. In contrast, maternal PCH (mat-PCH) is enriched for H3 lysine 9 tri-methylation (H3K9me3) and Hp1β. How PRC1 is targeted to pat-PCH, yet excluded from mat-PCH, has remained elusive. Here, we identify a PRC1 targeting mechanism that relies on Cbx2 and Hp1β. Cbx2 directs catalytically active PRC1 to PCH via its chromodomain (CDCbx2) and neighboring AT-hook (ATCbx2) binding to H3K27me3 and AT-rich major satellites, respectively. CDCbx2 prevents ATCbx2 from interacting with DNA at PCH marked by H3K9me3 and Hp1β. Loss-of-function studies show that Hp1β and not H3K9me3 prevents PRC1 targeting to mat-PCH. Our findings indicate that CDCbx2 and ATCbx2 separated by a short linker function together to integrate H3K9me3/HP1 and H3K27me3 states

Epigenetics controls of the cell cycle : functions and regulation of the lysine methyltransferase PR-Set7

La lysine méthyltransférase PR-Set7 est responsable de la monométhylation de la lysine 20 de l'histone H4 (H4K20me1). Son expression varie au cours du cycle cellulaire. D'un niveau peu élevé en phase S, l'enzyme atteint un niveau maximum au cours de la mitose. Mon projet de thèse avait pour but de caractériser les fonctions de PR-Set7 et les raisons de cette régulation au cours du cycle. Présentés sous forme de publication, les résultats de ma thèse montrent que PR-Set7 induit un signal H4K20me1 au niveau des origines de réplication pendant la mitose, ce qui permet le recrutement des complexes de pré-réplication (Pre-RC) contenant les facteurs nécessaires à la formation des fourches de réplication lors la phase S suivante. En effet, la présence de PR-Set7 sur une séquence d'ADN spécifique est suffisante pour induire le co-recrutement des protéines du complexe Pre-RC, tandis que l'inactivation de l'enzyme conduit au contraire à un défaut d'assemblage de ces complexes suivi d'un stress réplicatif. Lors de la phase S, PR-Set7 est dégradée par le complexe Cul4-DDB1, via son interaction avec la protéine PCNA. Cette dégradation permet la disparition du signal H4K20me1 des origines et l'inhibition des complexes Pre-RC, s'assurant ainsi que les origines sont actives une seule fois par cycle cellulaire. La mutation du domaine d'interaction avec PCNA est suffisante pour empêcher la dégradation de PR-Set7, entraînant alors la maintenance du signal H4K20me1 et une activation répétée des origines pendant la phase S (phénotype de sur-réplication). L'ensemble de mes résultats établissent PR-Set7 et le signal H4-K20me1 comme un nouveau mécanisme épigénétique de contrôle des origines de réplication chez les mammifères.The lysine methyltransferase PR-Set7 is responsible of the monomethylation of lysine 20 of histone H4 (H4K20me1). Its expression is cell-cycle regulated. With weak levels in S phase, this enzyme reach a peak level during mitosis. My PhD project was to characterize the functions of PR-Set7 and the reasons underlying its cell-cycle regulation. Presented as publications, my results show that PR-Set7 induces H4K20me1 on replication origins during mitosis, which allows recruitment of pre-replication complexes (Pre-RC) containing all the factors required to create replication forks during the next S phase. Indeed, the presence of PR-Set7 on a specific DNA sequence is sufficient to induce the co-recruitment of Pre-RC complex proteins, whereas the inactivation of this enzyme leads to defects in the assembly of these complexes followed by a replicative stress. During S phase, PR-Set7 is degraded par the Cul4-DDB1 complex through its association with PCN A. This degradation induces the disappearance of H4K20me1 on origins and inhibition of Pre-RC complexes, ensuring that origins are activated only once per cell cycle. Mutations in the interaction domain with PCNA are sufficient to prevent PR-Set7 degradation, leading to the maintenance of H4K20me1 and a multiple activation of origins during S phase (over-replication phenotype). My results establish PR-Set7 and H4K20me1 as a new epigenetic mechanism to control replication origins in mammals

Telomere chromatin establishment and its maintenance during mammalian development

International audienceTelomeres are specialized structures that evolved to protect the end of linear chromosomes from the action of the cell DNA damage machinery. They are composed of tandem arrays of repeated DNA sequences with a specific heterochromatic organization. The length of telomeric repeats is dynamically regulated and can be affected by changes in the telomere chromatin structure. When telomeres are not properly controlled, the resulting chromosomal alterations can induce genomic instability and ultimately the development of human diseases, such as cancer. Therefore, proper establishment, regulation, and maintenance of the telomere chromatin structure are required for cell homeostasis. Here, we review the current knowledge on telomeric chromatin dynamics during cell division and early development in mammals, and how its proper regulation safeguards genome stability

Polycomb function during oogenesis is essential for mouse embryonic development

In mammals, totipotent embryos are formed by fusion of highly differentiated gametes. Acquisition of totipotency concurs with chromatin remodeling of parental genomes, changes in the maternal transcriptome and proteome, and zygotic genome activation (ZGA). The inefficiency of reprogramming somatic nuclei in reproductive cloning suggests that intergenerational inheritance of germline chromatin contributes to developmental proficiency after natural conception. Here we show that Ring1 and Rnf2 , components of Polycomb-repressive complex 1 (PRC1), serve redundant transcriptional functions during oogenesis that are essential for proper ZGA, replication and cell cycle progression in early embryos, and development beyond the two-cell stage. Exchange of chromosomes between control and Ring1 / Rnf2 -deficient metaphase II oocytes reveal cytoplasmic and chromosome-based contributions by PRC1 to embryonic development. Our results strongly support a model in which Polycomb acts in the female germline to establish developmental competence for the following generation by silencing differentiation-inducing genes and defining appropriate chromatin states

Contrôles épigénétiques du cycle cellulaire (fonctions et régulation de la lysine méthyltransférase PR-Set7)

La lysine méthyltransférase PR-Set7 est responsable de la monométhylation de la lysine 20 de l'histone H4 (H4K20me1). Son expression varie au cours du cycle cellulaire. D'un niveau peu élevé en phase S, l'enzyme atteint un niveau maximum au cours de la mitose. Mon projet de thèse avait pour but de caractériser les fonctions de PR-Set7 et les raisons de cette régulation au cours du cycle. Présentés sous forme de publication, les résultats de ma thèse montrent que PR-Set7 induit un signal H4K20me1 au niveau des origines de réplication pendant la mitose, ce qui permet le recrutement des complexes de pré-réplication (Pre-RC) contenant les facteurs nécessaires à la formation des fourches de réplication lors la phase S suivante. En effet, la présence de PR-Set7 sur une séquence d'ADN spécifique est suffisante pour induire le co-recrutement des protéines du complexe Pre-RC, tandis que l'inactivation de l'enzyme conduit au contraire à un défaut d'assemblage de ces complexes suivi d'un stress réplicatif. Lors de la phase S, PR-Set7 est dégradée par le complexe Cul4-DDB1, via son interaction avec la protéine PCNA. Cette dégradation permet la disparition du signal H4K20me1 des origines et l'inhibition des complexes Pre-RC, s'assurant ainsi que les origines sont actives une seule fois par cycle cellulaire. La mutation du domaine d'interaction avec PCNA est suffisante pour empêcher la dégradation de PR-Set7, entraînant alors la maintenance du signal H4K20me1 et une activation répétée des origines pendant la phase S (phénotype de sur-réplication). L'ensemble de mes résultats établissent PR-Set7 et le signal H4-K20me1 comme un nouveau mécanisme épigénétique de contrôle des origines de réplication chez les mammifères.The lysine methyltransferase PR-Set7 is responsible of the monomethylation of lysine 20 of histone H4 (H4K20me1). Its expression is cell-cycle regulated. With weak levels in S phase, this enzyme reach a peak level during mitosis. My PhD project was to characterize the functions of PR-Set7 and the reasons underlying its cell-cycle regulation. Presented as publications, my results show that PR-Set7 induces H4K20me1 on replication origins during mitosis, which allows recruitment of pre-replication complexes (Pre-RC) containing all the factors required to create replication forks during the next S phase. Indeed, the presence of PR-Set7 on a specific DNA sequence is sufficient to induce the co-recruitment of Pre-RC complex proteins, whereas the inactivation of this enzyme leads to defects in the assembly of these complexes followed by a replicative stress. During S phase, PR-Set7 is degraded par the Cul4-DDB1 complex through its association with PCN A. This degradation induces the disappearance of H4K20me1 on origins and inhibition of Pre-RC complexes, ensuring that origins are activated only once per cell cycle. Mutations in the interaction domain with PCNA are sufficient to prevent PR-Set7 degradation, leading to the maintenance of H4K20me1 and a multiple activation of origins during S phase (over-replication phenotype). My results establish PR-Set7 and H4K20me1 as a new epigenetic mechanism to control replication origins in mammals.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

SUMO ylated PRC 1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin

Chromatin integrity is essential for cellular homeostasis. Polycombgroup proteins modulate chromatin states and transcriptionallyrepress developmental genes to maintain cell identity. They alsorepress repetitive sequences such as major satellites and consti-tute an alternative state of pericentromeric constitutive hete-rochromatin at paternal chromosomes (pat-PCH) in mouse pre-implantation embryos. Remarkably, pat-PCH contains the histoneH3.3 variant, which is absent from canonical PCH at maternal chro-mosomes, which is marked by histone H3 lysine 9 trimethylation(H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2-modified CBX2-containing Polycomb Repressive Complex 1 (PRC1)recruits the H3.3-specific chaperone DAXX to pat-PCH, enablingH3.3 incorporation at these loci. Deficiency of Daxx or PRC1 compo-nents Ring1 and Rnf2 abrogates H3.3 incorporation, induces chro-matin decompaction and breakage at PCH of exclusively paternalchromosomes, and causes their mis-segregation. Complementationassays show that DAXX-mediated H3.3 deposition is required forchromosome stability in early embryos. DAXX also regulates repres-sion of PRC1 target genes during oogenesis and early embryogene-sis. The study identifies a novel critical role for Polycomb inensuring heterochromatin integrity and chromosome stability inmouse early development

Cocktails of NSAIDs and 17α Ethinylestradiol at Environmentally Relevant Doses in Drinking Water Alter Puberty Onset in Mice Intergenerationally

Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinyl-estradiol (EE2) are among the most relevant endocrine-disrupting pharmaceuticals found in the environment, particularly in surface and drinking water due to their incomplete removal via wastewater treatment plants. Exposure of pregnant mice to NSAID therapeutic doses during the sex determination period has a negative impact on gonadal development and fertility in adults; however, the effects of their chronic exposure at lower doses are unknown. In this study, we investigated the impact of chronic exposure to a mixture containing ibuprofen, 2hydroxy-ibuprofen, diclofenac, and EE2 at two environmentally relevant doses (added to the drinking water from fetal life until puberty) on the reproductive tract in F1 exposed mice and their F2 offspring. In F1 animals, exposure delayed male puberty and accelerated female puberty. In post-pubertal F1 testes and ovaries, differentiation/maturation of the different gonad cell types was altered, and some of these modifications were observed also in the non-exposed F2 generation. Transcriptomic analysis of post-pubertal testes and ovaries of F1 (exposed) and F2 animals revealed significant changes in gene expression profiles and enriched pathways, particularly the inflammasome, metabolism and extracellular matrix pathways, compared with controls (non-exposed). This suggested that exposure to these drug cocktails has an intergenerational impact. The identified Adverse Outcome Pathway (AOP) networks for NSAIDs and EE2, at doses that are relevant to everyday human exposure, will improve the AOP network of the human reproductive system development concerning endocrine disruptor chemicals. It may serve to identify other putative endocrine disruptors for mammalian species based on the expression of biomarkers.</jats:p