Search

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent inMus spretuswhich can interbreed withMus musculusbut lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

Repeat element transcription plays a vital role in early embryonic development. The expression of repeats such as MERVL characterises mouse embryos at the 2‐cell stage and defines a 2‐cell‐like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of major satellite repeats and MERVL transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de‐repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2‐cell state, shifting the balance between the stem cell and 2‐cell‐like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor., The forkhead domain transcription factor FOXD3 is involved in the heterochromatin‐mediated repression of major satellite repeats and MERVL, thereby counteracting the transcription of 2‐cell state promoting genes in mouse embryonic stem cells.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity., Histone H3K9 methylation (H3K9me) states define repressed chromatin in eukaryotic cells. Here the authors reveal complete loss of all H3K9me in mammalian cells through successive deletion of H3K9 methyltransferase genes that results in the dissolution of heterochromatin and the derepression of nearly all repeat families.

Reversing chromatin dynamics for development Compacted chromatin regions, marked by trimethylation of histone H3 at position lysine 9 (H3K9me3), occur at highly repeated DNA sequences, helping to suppress recombination and gene expression. Because pluripotent cells contain low levels of H3K9me3 heterochromatin relative to differentiated cells, it has been thought that an increase in such heterochromatin helps to define cell differentiation. Nicetto et al. used two independent methods to examine compacted heterochromatic domains and found that H3K9me3 compaction increased at protein-coding genes during early mouse organogenesis. During differentiation, these domains open up to allow cell-specific expression. Loss of heterochromatin by genetic inactivation of the H3K9me3 methyltransferases caused ectopic expression of cell-inappropriate genes and tissue pathology. Science , this issue p. 294

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner., Author summary While RNAi has been recognized as an efficient antiviral defense system in organisms such as plants and insects, its physiological importance in mammals remains to be determined. DICER is an enzyme involved in cleaving long double-stranded RNAs and is essential for RNAi induction. Using mass spectrometry analysis, we determined its interactome in human cells and showed that RNA binding proteins such as PKR are specifically enriched upon infection with the Sindbis virus or the Semliki forest virus. We determined that the N terminal helicase domain of the DICER protein acts as a platform to recruit these factors during infection and that its deletion confers an antiviral activity to DICER.

Repeat element transcription plays a vital role in early embryonic development. Expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage, and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of repeat element transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2-cell like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.

RNA silencing is a small RNA based mechanism regulating gene expression and involved in many biological processes in most eukaryotes. In plants, nematodes and arthropods, this mechanism participates to antiviral defense. In mammals, although the RNA silencing machinery is present and needed for the microRNA pathway, its importance as an antiviral defense is still debated. In recent years, several studies have attempted to answer to the question of whether RNA silencing as an antiviral pathway is retained in mammals. However, these studies did not provide a clear answer yet. In this review, we will present the arguments for and against a relevant antiviral role of RNA interference (RNAi) in mammals, by discussing examples of active and functional mammalian antiviral RNAi in specific cell types and/or in specific conditions.

Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein‐level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double‐stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA‐induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth., AGO1x, a translational readthrough isoform of Argonaute 1, localizes to the nucleus and interacts with proteins involved in dsRNA processing, promoting dsRNA clearance.

Double-stranded RNA binding (DRB) proteins are generally considered as promoting cofactors of Dicer or Dicer-like (DCL) proteins that ensure efficient and precise production of small RNAs, the sequence-specificity guide of RNA silencing processes in both plants and animals. However, the characterization of a new clade of DRB proteins in Arabidopsis has recently challenged this view by showing that DRBs can also act as potent inhibitors of DCL processing. This is achieved through sequestration of a specific class of small RNA precursors, the endogenous inverted-repeat (endoIR) dsRNAs, thereby selectively preventing production of their associated small RNAs, the endoIR-siRNAs. Here, we concisely summarize the main findings obtained from the characterization of these new DRB proteins and discuss how the existence of such complexes can support a potential, yet still elusive, biological function of plant endoIR-siRNAs.

International audience; In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

Copy number variations are genomic structural variants that are frequently associated with human diseases. Among these copy number variations, duplications of DNA segments are often assumed to lead to dosage effects by increasing the copy number of either genes or their regulatory elements. We produced a series of large targeted duplications within a conserved gene desert upstream of the murine HoxD locus. This DNA region, syntenic to human 2q31-32, contains a range of regulatory elements required for Hoxd gene transcription, and it is often disrupted and/or reorganized in human genetic conditions collectively known as the 2q31 syndrome. Unexpectedly, one such duplication led to a transcriptional down-regulation in developing digits by impairing physical interactions between the target genes and their upstream regulatory elements, thus phenocopying the effect obtained when these enhancer sequences are deleted. These results illustrate the detrimental consequences of interrupting highly conserved regulatory landscapes and reveal a mechanism where genomic duplications lead to partial loss of function of nearby located genes.

SummaryThe evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a “regulatory archipelago,” provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.PaperFlick

Hox genes are required during the morphogenesis of both vertebrate digits and external genitals. We investigated whether transcription in such distinct contexts involves a shared enhancer-containing landscape. We show that the same regulatory topology is used, yet with some tissue-specific enhancer-promoter interactions, suggesting the hijacking of a regulatory backbone from one context to the other. In addition, comparable organizations are observed at both HoxA and HoxD clusters, which separated through genome duplication in an ancestral invertebrate animal. We propose that this convergent regulatory evolution was triggered by the preexistence of some chromatin architecture, thus facilitating the subsequent recruitment of the appropriate transcription factors. Such regulatory topologies may have both favored and constrained the evolution of pleiotropic developmental loci in vertebrates.

SummaryCCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.

Hox genes are critical regulators of embryonic development in bilaterian animals. They exhibit a unique mode of transcriptional regulation where the position of the genes along the chromosome corresponds to the time and place of their expression during development. The sequential temporal activation of these genes in the primitive streak helps determining their subsequent pattern of expression along the anterior-posterior axis of the embryo, yet the precise correspondence between these two collinear processes is not fully understood. In addition, vertebrate Hox genes evolved similar modes of regulation along secondary body axes, such as the developing limbs. We review the current understanding of the mechanisms operating during activation, maintenance and silencing of Hox gene expression in these various contexts, and discuss the evolutionary significance of their genomic organization.

Collinearity Cracked in Tetrapod Limbs During limb development, the time a nd place of Hox transcription are fixed by respective gene position within the gene cluster. Andrey et al. (p. 1234167 ; see the Perspective by Rodrigues and Tabin ) found that this enigmatic property results from the opposite and successive actions of two large regulatory landscapes located on either side of the mouse Hox locus. In the early phase, one of these topological domains regulates transcription in the proximal limb until a switch occurs toward the other topological domain, which takes over the regulation in the distally developing digits. As a side effect of this antagonistic regulatory strategy, cells in-between have lessened Hox transcription, which generates the wrist.

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.

Collinear expression of 5′ Hoxd genes during mammalian limb development is required to properly determine digits number and identity. We recently proposed a two-step model to account for the underlying regulatory mechanism, which involves an initial looping and recognition of the cluster by a complex of enhancer sequences, followed by a micro-scanning of nearby-located genes. This model could account for both the wild-type dosage of the various Hoxd transcripts, as well as for their quantitative variations in several mutant strains. Here, using the Chromosome Conformation Capture (3C) technique, we confirm the initial looping step by showing that the 5′ extremity of the HoxD cluster physically interacts with two previously identified, remote regulatory sequences displaying enhancer activity in developing digits. This spatial conformation is specific to presumptive digits and is not observed in tissues devoid of Hox gene transcription. The flexibility of these long-range interactions is illustrated by the ability of the enhancers to activate reporter transgenes inserted at various positions along the locus. To further define this regulatory landscape, we produced a large chromosomal inversion, separating the reporter transgene from the digit enhancers and HoxD cluster. Surprisingly, this configuration fails to abolish the transgene expression in developing digits, but leads to a major down-regulation of Hoxd genes in this domain, and concurrent morphological defects. These results suggest that the transcriptional activation of Hoxd genes requires the activity of additional and distant regulatory sequences, in addition to the enhancers identified so far by transgenic analysis.

During the development of mammalian digits, clustered Hoxd genes are expressed following a collinear regulatory strategy, leading to both the growth of digits and their morphological identities. Because gene dosage is a key parameter in this system, we used a quantitative approach, associated with a collection of mutant stocks, to investigate the nature of the underlying regulatory mechanism(s). In parallel, we elaborated a mathematical model of quantitative collinearity, which was progressively challenged and validated by the experimental approach. This combined effort suggested a two-step mechanism, which involves initially the looping and recognition of the cluster by a complex including two enhancer sequences, followed by a second step of microscanning of genes located nearby. In this scenario, the respective rank of the genes, with respect to the 5′ extremity of the cluster, is primordial, as well as different gene-specific affinities. This model accounts for the quantitative variations observed in our many mutant strains, and reveals the molecular constraint leading to thumbness; i.e., why a morphological difference must occur between the most anterior digit and the others. We also show that the same model applies to the collinear regulation of Hox genes during the emergence of external genitalia, though with some differences likely illustrating the distinct functionalities of these structures in adults.

Mesomelic dysplasia is a severe shortening of forearms and forelegs, and is found in several distinct human syndromes. Here, we report the cloning of the breakpoints of a human t(2;8)(q31;p21) balanced translocation associated with mesomelic dysplasia of the upper limbs, as well as with vertebral defects. We show that this translocation does not disrupt any gene, hence it most likely exerts its deleterious effect by modifying gene regulation. The HOXD complex lies approximately 60 kb from the translocation breakpoint on chromosome 2. This cluster of genes has an important role in the development of both the vertebral column and the limbs. Only a few cases of mutations of these homeotic genes have been described so far in humans. However, gain- and loss-of-function of Hoxd genes in mice can induce mesomelic dysplasia-like phenotypes, suggesting that misexpression of HOXD genes may indeed be at the origin of this hereditary phenotype.

SummaryHox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common “budding toolkit” between the cecum and the limbs.

Vertebrate genes controlling critical developmental processes are often regulated by complex sets of global enhancer sequences, located at a distance, within neighboring gene deserts. Recent technological advances have made it possible to investigate the spatial organization of these 'regulatory landscapes'. The integration of such datasets with information on chromatin status, transcriptional activity and nuclear localization of these loci, as well as the effects of genetic modifications thereof, may bring a more comprehensive understanding of tissue- and/or stage-specific gene regulation in both normal and pathological contexts. Here, we review the impact of recent technological advances on our understanding of large-scale gene regulation in vertebrates, by focusing on paradigmatic gene loci.

During development, a properly coordinated expression ofHoxgenes, within their different genomic clusters is critical for patterning the body plans of many animals with a bilateral symmetry. The fascinating correspondence between the topological organization ofHoxclusters and their transcriptional activation in space and time has served as a paradigm for understanding the relationships between genome structure and function. Here, we review some recent observations, which revealed highly dynamic changes in the structure of chromatin atHoxclusters, in parallel with their activation during embryonic development. We discuss the relevance of these findings for our understanding of large-scale gene regulation.