Readers of the m6A epitranscriptomic code

International audienceN6-methyl adenosine (m6A) is the most prevalent and evolutionarily conserved, modification of polymerase II transcribed RNAs. By post-transcriptionally controlling patterns of gene expression, m6A deposition is crucial for organism reproduction, development and likely stress responses. m6A mostly mediates its effect by recruiting reader proteins that either directly accommodate the modified residue in a hydrophobic pocket formed by their YTH domain, or otherwise have their affinity positively influenced by the presence of m6A. We firstly describe here the evolutionary history, and review known molecular and physiological roles of eukaryote YTH readers. In the second part, we present non YTH-proteins whose roles as m6A readers largely remain to be explored. The diversity and multiplicity of m6A readers together with the possibility to regulate their expression and function in response to various cues, offers a multitude of possible combinations to rapidly and finely tune gene expression patterns and hence cellular plasticity

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods

CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells

Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

Etude des mécanismes de formation et d organisation de l hétérochromatine chez la souris

La molécule d ADN porte l ensemble des informations génétiques nécessaires au fonctionnement de chaque cellule. Cette molécule est compactée dans le noyau grâce aux histones ce qui forme la chromatine. La chromatine peut être plus ou moins compactée. Ces différents états de compaction participent à la régulation de l expression génique dans les cellules. Par exemple la chromatine décondensée, l euchromatine, corrèle avec une chromatine active tandis que la chromatine condensée, l hétérochromatine, est réprimée. Pendant ma thèse, je me suis intéressée aux mécanismes impliqués dans la formation et l organisation de l hétérochromatine chez la souris. D une part, nous avons identifié les marques biochimiques qui caractérisent l hétérochromatine au niveau du gène Necdin qui appartient à une région d hétérochromatine facultative. Nous avons mis en évidence que les histones de cette région sont enrichies en une marque, la triméthylation de la lysine 20 de l histone H4, qui n avait pas encore été identifiée dans une région d hétérochromatine facultative. Nous avons également mis en évidence que la présence de cette marque corrèle avec une structure de chromatine condensée et inaccessible aux protéines liant l ADN. D autre part, nous avons analysé le rôle d un facteur d assemblage de la chromatine, CAF-1, chez la souris et en cellules souches embryonnaires (ES). Nous avons montré que CAF-1 est un facteur essentiel pour le développement de la souris et pour la survie des cellules ES. Nous avons également mis en évidence qu en l absence de ce facteur, les chromocentres et l hétérochromatine péricentrique sont désorganisés.In eukaryotes, DNA is packaged with histones proteins into chromatin, compacting DNA some 10 000 fold. The nucleosome is the fundamental unit of chromatin and is composed of an octamer of the four core histones (H2A, H2B, H3, H4) around which 147 base paires of DNA are wrapped. There are two major types of chromatin : euchromatin and heterochromatin. Euchromatin generally keeps genes competent for transcription whereas heterochromatin contains predominantly transcriptionally silent genes and includes specialized chromosomes structures such as centromeres and telomeres. We have tried to unravel the mechanisms that govern heterochromatin formation and organization in the mouse. First, we studied the formation of the facultative heterochromatin in the Prader-Willi Syndrome region. The maternal allele of the genes in this region are repressed while the paternal ones are imprinted. We found that this heterochromatin differs biochemically from the facultative heterochromatin found on the inactive X chromosome in the mammalian females. Indeed, the chromatin of these genes is enriched in a post-translation histone marks modification previously found in centromeric chromatin. We also showed that this chromatin is more condensed and less accessible to nucleases than usual facultative heterochromatin. Second, we have studied the role of the chromatin assembly factor 1, CAF-1, in the developing mouse and embryonic stem cells. We found that CAF-1 is an essential gene for mouse development. Our data also show that CAF-1 is required for the correct organization of the pericentric heterochromatin in mouse embryo and embryonic stem cells.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells

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Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of <i>HoxA</i> Genes in Developing Limbs

<div><p><i>HoxA</i> genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of <i>bona fide</i> transcriptional enhancers controlling <i>HoxA</i> genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with <i>HoxA</i> genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of <i>HoxA</i> and <i>HoxD</i> regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.</p></div

Extensive clustering of genes and enhancers highlights a complex regulation network in distal limbs.

<p><b>A,B.</b> 5C interaction matrix of the <i>HoxA</i> cluster and its upstream regulatory region in distal limb (<b>A</b>) and head (<b>B</b>). The 5C data was generated by 5C-seq using tissues from E12.5 embryos, and is presented in the form of heatmaps according to color scales as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen-1004018-g002" target="_blank">Figure 2</a>. Heatmaps above the linear diagram of the genomic region show interaction frequencies for each restriction fragment, irrespective of their size. Heatmaps at the bottom show the mean interaction frequencies per 20 kb DNA fragment and were obtained from binning and smoothing of the 5C raw data. Expressed genes within the region are colored in red. The yellow and green shading links the genomic position of <i>HoxA</i> and <i>Evx1</i> genes, and the enhancer clusters to the corresponding areas in heatmaps. Black arrows point to interactions between the gene sub-TADs and enhancer sub-TADs. White lines delineate the TAD and sub-TADs therein. Dashed white lines are drawn to highlight the sub-TAD interactions. <b>C.</b> Topological organization of the <i>HoxA</i> cluster and <i>Evx1</i>. Genes are organized in three sub-TADs in the limb (<i>top</i>). Interaction enrichment in head tissues compared to the limb (<i>bottom</i>) shows significant increase in interaction between the gene sub-TADs in the head. Smoothing was performed based on distance (8 kb) and heatmap intensities represent the mean of interaction frequency for each 8 kb window. <b>D.</b> Extensive limb-enriched interactions between distal <i>HoxA</i> enhancers suggest that a physical network regulates 5′ <i>HoxA</i> genes in the limb. The interaction matrix of the region containing enhancer e10 to e18 is shown in the form of a heatmap. Limb-enriched contacts are shown in red according to the color scale as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen-1004018-g002" target="_blank">Figure 2</a>.</p

Candidate limb enhancers reside on the telomeric side of the <i>HoxA</i> cluster.

<p><b>A, B.</b> Distal limb enhancer activity lies upstream of the <i>HoxA</i> cluster and does not require sequences within it. Expression of the Neomycin and Hygromycin reporter genes flanking the cluster were analyzed by whole mount <i>in situ</i> hybridization on E11.5 embryos. In embryos where the <i>HoxA</i> cluster is intact (<b>A</b>), expression of the upstream Hygromycin reporter was detected in the distal part of the limb while downstream neomycin transcripts were not. TKNeo: minimal thymidine kinase promoter upstream of Neomycin reporter gene. PGKHygro: minimal phosphoglycerate kinase-1 promoter upstream of Hygromycin reporter gene. Arrow above the <i>HoxA</i> cluster diagram shows transcription direction. <b>B.</b> Neomycin expression after deletion of the cluster and PGKHygro by recombination of <i>loxP</i> sites flanking the reporter genes shows that sequences within the cluster are not required for distal limb enhancer activity. <b>C.</b> Distal limb cells analyzed in this study express 5′ <i>HoxA</i> genes (<i>Hoxa9</i> to <i>a13</i>). <i>HoxA</i> gene expression in developing limbs is illustrated on the <i>left</i>. The dotted line indicates the area micro-dissected to collect distal limb cells for analysis. Stylopod: upper arm, zeugopod: lower arm, mesopod: wrist, autopod: hand. <b>D.</b> The position of candidate enhancer sequences was identified by ChIP-seq. Proteins known as being enriched at active enhancers (RNAP2, Med12, p300) and the H3K27Ac histone mark was examined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#s4" target="_blank">Materials and Methods</a>. The y-axis corresponds to “reads per million” except for the p300 data where the number of sequence reads is shown. Colored rectangles below each track indicate the position of significant peaks. The position of candidate enhancers (e1 to e19) is highlighted in green below the genomic region characterized, where transcriptionally active genes are in red and arrows indicate transcription direction. Sequence conservation in the chicken is shown on the bottom.</p