Polycomb Mediated Epigenetic Silencing and Replication Timing at the INK4a/ARF Locus during Senescence

International audienceBACKGROUND: The INK4/ARF locus encodes three tumor suppressor genes (p15(Ink4b), Arf and p16(Ink4a)) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized. PRINCIPAL FINDINGS: Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence. CONCLUSIONS: We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus

Personalized dietary advices provided by a dietitian increase calcium intake in outpatients with multiple sclerosis—Results from a randomized, controlled, single-blind trial

Background and aimsMultiple sclerosis (MS) is associated with osteoporosis, possibly due to neurological disability and decreased calcium intake. The objective of this study was to evaluate the efficacy of a personalized nutritional advice program by a dietitian compared to the delivery of a standard advice form to optimize dietary calcium intake in outpatients with MS.MethodsWe performed a randomized, controlled, parallel trial comparing the efficacy of a personalized dietary advice (PDA) program to standard advice form (SAF) to increase daily calcium intake in MS patients. The study population was composed by patients with relapsing-remitting MS aged 18–69 years old. PDA program consisted in dietary advice delivered by a dietitian at baseline, 1 month, and 3 months. Calcium and nutrient intake in patients from both groups was evaluated at baseline and 6 months using a dietary survey.ResultsOf the 194 patients screened for inclusion, 182 patients were included (79% female, median age of 42 years, and median EDSS of 2.0), and randomized to SAF (n = 92) or PDA (n = 90). At 6 months, median calcium intake increased by 241 mg/day in the PDA group and decreased by 120 mg/day in the SAF group (p < 0.0001). However, the median calcium intake was 947 mg/day in the SAF group and 778 mg/day in the PDA group at baseline (p = 0.0077), potentially favoring the effect of dietary advice. Complementary analyses focusing on patients with insufficient calcium intakes at baseline revealed comparable values in both groups (p = 0.69). Of those, patients included in the PDA group obtained significantly higher calcium intakes at 6 months than patients from the SAF group (p = 0.0086) independently of EDSS, PASAT, HADS and EQ-5D scores.ConclusionThis work shows the efficacy of dietary management based on personalized advice program over 3 months to durably increase calcium consumption in MS patients with insufficient calcium intake.Clinical trial registrationclinicaltrials.gov, identifier NCT02664623

Rôle des protéines polycomb et trithorax dans le contrôle épigénétique des gènes cibles

Mon travail de thèse s'est axé sur la compréhension des mécanismes de régulation épigénétique de la transcription des gènes cibles des protéines des groupes trithorax et polycomb. Ces protéines hautement conservées au cours de l'évolution interviennent notamment au cours du développement embryonnaire, dans la prolifération cellulaire et la " mémoire cellulaire ". Elles régulent la transcription de leurs gènes cibles via l'établissement et l'interprétation de modifications épigénétiques qui conduisent au remodelage de la structure de la chromatine. Mon premier projet a porté sur l'étude du domaine SET de la protéine trithorax, MLL1. Ce domaine confère son activité méthyltransférase qui est essentielle à sa fonction dans la régulation épigénétique des gènes. Ce travail démontre in vivo l'importance capitale du domaine SET dans les mécanismes épigénétiques de maintien de l'expression des gènes Hox par la protéine MLL1 au cours du développement et révèle l'existence d'un lien entre la méthylation des histones et la méthylation de l'ADN qui dépendent de MLL1 aux gènes cibles. Au cours de mon deuxième projet de thèse j'ai étudié le rôle des protéines polycomb et trithorax dans la régulation du locus suppresseur de tumeur INK4/ARF. Ce locus code pour deux protéines p16INK4a et p19ARF capables d'induire la sénescence. Mes travaux montrent que le locus INKa/ARF est directement régulé par les protéines polycomb et la protéine MLL1 via l'établissement de modifications épigénétiques et que les protéines polycomb pourraient moduler le timing de réplication du locus ce qui constituerait un autre mécanisme de régulation.During my thesis my work was focused on the understanding of the epigenetic mechanisms of gene regulation by the polycomb and trithorax group proteins. These proteins are highly conserved during evolution and are implicated notably in cell proliferation and epigenetic memory during development. Polycomb and trithorax proteins regulate their target genes by establishing and interpreting epigenetic modifications leading to chromayin structure remodeling. During my first project, I studied the fuction of the SET domain of the trithorax, protein, MLL1. I could demonstrate in vivo that the SET domain is critical for MLL function. The SET domain is essential to sustain the expression of selected Hox genes duringembryonic development. These obsevations provide previously undescribed for the in vivo relationship and SET domain dependence between histone methylation and DNA methylation on MLL target genes.In the second part of my thesis, I wanted to determine the molecular mechanims of INK4a/ARF regulation by polycomb protein. I demonstrated that polycomb proteins and MLL regulate directly theINK4a/ARF locus. Moreover, I could show that polycomb proteins contronl the replication timing of the INK4a/ARF locus during senescence xhich would constitute a new mechanism of INK4a/ARF regulation by polycomb proteins.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

[Cellular ageing, ageing and cancer]

National audienceCellular division is essential for the survival of the multicellular organisms which contain renewable tissues. However, cellular division also puts the organisms in danger to develop any types of cancers. Cellular senescence has emerged in part as a tumor suppressor mechanism. Here we discuss the function and regulation of the tumor suppressor proteins INK4a/ARF in connection with replicative senescence, cancer and aging. double dagger

Vieillissement cellulaire, vieillissement et cancer

La division cellulaire est essentielle pour la survie des organismes multicellulaires qui contiennent des tissus renouvelables. Cependant, la division cellulaire met aussi les organismes en danger de développer tous types de cancers. La sénescence cellulaire est en partie maintenant considérée comme un mécanisme anti-tumoral. Nous discuterons ici de la fonction et de la régulation des protéines suppresseurs de tumeurs INK4a/ARF en relation avec la sénescence réplicative, le cancer et le vieillissement

Loss of EZH2 binding and H3K27me3 methylation at the <i>INK4a/ARF</i> locus during senescence.

<p>P3 proliferating and P10–12 senescent MEFs were subjected to ChIP assays using the indicated antibodies.</p

Analysis of Polycomb EZH2, M33 and BMI1 binding at the <i>INK4a/ARF</i> region.

<p>A) qPCR analysis of p16^INK4a and p19^ARF in young (P3), senescent (P10) and in <i>Bmi1</i> and <i>M33</i> mutant MEFs. B) B-galactosidase staining to detect senescent cells at passage 3 (P3) and passage 10 (P10). C) qPCR analysis of the mRNA levels of Polycomb EZH2 and Bmi1 in the indicated cells. D–F) Schematic diagram of the <i>INK4a/ARF</i> locus: amplified regions that were tested in ChIP experiments are indicated by red bars (sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#pone-0005622-t001" target="_blank">Table 1</a>). Wild type P3 (young), P10–12 (senescent), M33−/− (P4) and Bmi−/− (P4) MEFs were subjected to ChIP assays using anti EZH2, BMI1 and M33 antibodies. DNA enrichment was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#s2" target="_blank">Materials and Methods</a>. Bars represent the mean+/−s.d. of quantifications from two to four separate immunoprecipitations analyzed in triplicate.</p

BMI1 interacts with CDC6 and is required for CDC6 repressing function.

<p>A) Western Blot analysis of Myc-CDC6 Wild type and <i>Bmi1</i> mutant transduced cells. The antibodies used are indicated. GAPDH antibody is used as a loading control. Ab) quantitative PCR experiment showing p16^INK4a and p19^ARF expression in Myc-CDC6 transduced <i>Bmi1</i> mutant cells. B) CDC6 interacts specifically with BMI1: HA immunoprecipitated proteins extracted from HA-CDC6 transfected cells were separated by SDS-PAGE and immunoblotted with a BMI1 antibody. C) anti-Bmi1 immunoprecipitated proteins extracted from wild type thymocytes and immunoblotted with a CDC6 antibody. D) BMI1 is required for INK4a CDC6 mediated repression. Wild type MEFs transfected with Myc-CDC6 were immunostained with a specific antibody against p16^INK4a (red) and CDC6 (green) middle panel. Untransfected cells are shown on the upper panel. <i>Bmi1</i> mutant cells transfected with Myc-CDC6 (green) express high level of p16 (red) (Bottom panel).</p

Model for Pc-G and MLL1 proteins in regulation of cellular senescence at the <i>INK4a/ARF</i> locus.

<p>(A) In young proliferating cells, the PRC2 complex is bound at RD and at the <i>INK4a/ARF</i> locus and maintains the levels of H3K27me3. This allows the association of M33 and BMI1-containing PRC1 complex and repression of the INK4a/ARF genes. (B) In senescent or Polycomb mutant cells binding of EZH2 is lost, leading to the disruption of the PRC2 complex, the loss of H3K27me3 and to the recruitment of the MLL1 protein. We propose a model in which Polycomb/MLL1 and JMJD3 epigenetic modifications at the RD element impact the replication timing and the expression of the locus. Moreover, in senescent cells BMI1 binding is specifically lost at the RD element.</p

<i>INK4a/ARF</i> timing of replication.

<p>PCR based analysis of replication timing of the <i>INK4a/ARF</i> locus (exon 1b). BrdU pulse labeled cells were stained for DNA content with propidium iodide and sorted by flow cytometry into 5 cell cycle fractions (G1, S1, S2, S3 and G2M) according to DNA content. The Gbe D. melanogaster gene provides a control for recovery of BrdU-labeled DNA.</p