Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

In human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.We show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.We demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription

Disctincts roles for different formes of H3K4 methylation in two transcriptional repression mechanisms and discovery of a new molecular surveillance pathway linked to an excess of free histones

Les relations entre les histones qui composent les nucléosomes et le processus de transcription des gènes codants, sont à la fois multiples et extrêmement complexes. Au cours de ma thèse, je me suis intéressé à deux de ces relations. Tout d abord, une première étude a été réalisée en collaboration avec les laboratoires de Franck Holstege et de Catherine Dargemont. Ce travail permet de préciser clairement l effet des différentes formes de méthylation de la lysine 4 de l histone H3 sur l activité transcriptionnelle. Dans cette étude nous démontrons que la méthylation de H3K4 n influence la transcription que d un nombre très limité de gènes. Concernant ces gènes, un profil non conventionnel de distribution des formes de méthylation de H3K4 a été identifié par la présence inhabituelle d un enrichissement en 3 de ces gène des formes di- et triméthylées de H3K4.L effet majoritaire de cette marque est d induire une répression transcriptionnelle selon au moins deux mécanismes distincts. L enrichissement atypique de la triméthylation de H3K4 influence négativement l expression des gènes via la production d ARN non codant anti-sens. Concernant l effet répressif associé à la diméthylation de H3K4, la quantité d ARN anti-sens ainsi que sa production ne sont pas impliquées.Dans une seconde étude réalisée en collaboration avec les laboratoires de Sebastian Chavez etd Akash Gunjan, nous nous sommes intéressés au complexe FACT qui est impliqué dans l assemblage et le désassemblage des nucléosomes lors du passage de l ARN polymérase II. Jusqu alors, un défaut de croissance chez les mutants thermosensibles du complexe FACT avait pu être observé. Dans notre étude, nous montrons que l altération de FACT conduit, lors de la transcription, à l éviction d histones normalement incorporées à la chromatine. L accumulation de ces histones libres à fort potentiel toxique, induit une répression spécifique de CLN3 qui code pour la première cycline dephase G1. Pour la première fois, nous mettons en évidence dans cette étude l existence d un mécanisme de surveillance moléculaire du cycle cellulaire induit par l excès d histones non incorporées à la chromatineRelationships between histones, components of nucleosomes, and the transcription process of coding genes are both multiple and extremely complex. During my Thesis, I looked at twoof these relationships. First, we performed a study in collaboration with the Franck Holstedge and Catherine Dargemont labs. This work has allowed us to clearly define the effect of various methylation forms of the lysine 4 of the histone 3 on gene transcription. In this study we have shownthat H3K4 methylation influences the transcription of only a very limited number of genes. For these genes, a non conventional distribution profile of H3K4 methylation forms has been identified by the presence of an unusual enrichment in di- and trimethylated H3K4 in the 3 of these genes. The principal effect of this mark is to promote transcriptional repression by at least two distinct mechanisms. The atypical enrichment of H3K4 trimethylation negatively influences gene expressionvia the production of non coding antisense RNA. For the repressive effect associated with dimethylH3K4, the quantity of antisense RNA as well as its production are not involved. We propose severalh ypotheses that link our results to the data known on this subject. In a second study performed incollaboration with the Sebastian Chavez and Akash Gunjan labs, we concentrated on the FACT complex that is involved in the assembly and disassembly of nucleosomes as RNA polymerase IImoves past. Previously, a growth defect in thermosensitive mutants of the FACT complex had been observed. In our study, we show that FACT deterioration leads to the eviction of histones that arenormally incorporated into chromatin during transcription. The accumulation of these free histones,which have a high toxic potential, induces the specific repression of CLN3 which encodes for the firstcyclin of G1 phase. For the first time, we show in this study the existence of a cell cycle molecular surveillance mechanism that is induced by an excess of free histonesAIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Set1 represses sense transcription through promotion of antisense transcription.

<p>(A) Scheme showing <i>YGR110W</i> and <i>YGR111W</i> genes before (YGR) and after (YGR-ingdel) deletion of their intergenic region. The sense <i>YGR110W</i> transcript (<i>sYGR110W</i>), as well as the longer sense transcript in the YGR-ingdel strain are shown in dark grey, while the antisense transcript (SUT557) is shown in light grey. The position and 5′-3′ direction of the strand-specific probes used to detect the transcripts are also shown. (B) Autoradiographs of Northern blots hybridized with the strand-specific DNA probes designed to detect the sense (<i>sYGR110W</i>) or antisense (<i>asYGR110W</i>) transcripts of <i>YGR110W</i> in the YGR and YGR-ingdel strains with wild-type (<i>SET1</i>) or deleted <i>SET1</i> (<i>set1Δ</i>). An autoradiograph of the same blot hybridized with a tubulin probe (<i>TUB1</i>) was used as loading control. Quantitation of the bands are shown below each panel relative to the wt (SET1 YGR) strain for TUB1 and relative to the wt (SET1 YGR) strain and the loading control for the <i>sYGR110W</i> and <i>asYGR110W</i> panels.</p

Loss of H3K4 di- and trimethylation leads to a delay in repression kinetics for a subset of genes.

<p>(A) Hierarchical clustering of all genes with significant changes in mRNA expression during the shift from low to high glucose in any of the wt, <i>spp1</i>Δ and <i>set1</i>Δ time-courses. The log₂ values correspond to the difference with the zero time point of each time-course. (B) Hierarchical clustering of genes with delayed repression compared to wt. These genes were identified based on statistically significant differences between the mutant and wt time-courses (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#s4" target="_blank">Materials and Methods</a>). The first three panels show the differences in expression versus the wt zero time point. The last two panels (<i>spp1</i>Δ <i>vs wt</i>) and (<i>set1</i>Δ <i>vs wt</i>) depict the differences between mutant and wt for each different time point, by subtracting the log base 2 gene expression ratios of the wt time-course from the mutant time-course. (C) Hierarchical clustering of genes that show delay in activation.</p

COMPASS-repressed genes are derepressed upon decrease in nucleosome content.

<p>(A) The cellular expression level of H3 was analyzed by Western blotting in wt and <i>hht1</i>Δ <i>hhf1</i>Δ cells. (B) Correlation of the effects, as measured by log base 2 ratios, of low nucleosomes levels (<i>hht1</i>Δ <i>hhf1</i>Δ) and <i>SET1</i> deletion (<i>set1</i>Δ) on the COMPASS-repressed genes versus wt cells. <i>LYS2</i> is excluded as it is used as an auxotrophic marker for the <i>hht1</i>Δ <i>hhf1</i>Δ strain. <i>PHO84</i> is marked, as it is significantly repressed in the low nucleosome strain. The two histone genes are also depicted verifying the expected 2-fold reduction in their mRNA levels. (C) Strand-specific qPCR analysis, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen-1002952-g004" target="_blank">Figure 4B</a>, for the indicated backgrounds.</p

Loss of H3K4 di- and trimethylation results in upregulation of a subset of genes.

<p>(A) Commassie-stained gel of purified histones from the indicated strains (top) and western blots with antibodies directed against H3 carboxy-terminus and the different H3K4 methylation states (bottom). (B) Hierarchical clustering of all genes with significantly changed mRNA expression (p-value less than 0.01 and fold-change versus wild-type more than 1.7) in at least two COMPASS mutants. Fold-change of mRNA expression in mutant versus wild-type is indicated by the colour bar as log₂ values. Number of genes below each heatmap correspond to the genes called significant in each mutant. (C) Genes depicted in the same order as in B for the H3K4R point mutant and <i>bre1</i>Δ.</p