Replication Fork Polarity Gradients Revealed by Megabase-Sized U-Shaped Replication Timing Domains in Human Cell Lines

In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome

Open for connections: HiCAR reveals the interactions of accessible DNA

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Detection of Allele-Specific 3D Chromatin Interactions Using High-Resolution In-Nucleus 4C-seq

International audienceChromosome Conformation Capture techniques are a set of methods used to determine 3D genome organization through the capture and identification of physical contacts between pairs of genomic loci. Among them, 4C-seq (Circular Chromosome Conformation Capture coupled to high-throughput sequencing) allows the identification and quantification of the sequences interacting with a pre-selected locus of interest. 4C-seq has been widely used in the literature, mainly to study chromatin loops between enhancers and promoters or between CTCF binding sites and to identify chromatin domain boundaries. As 3D-contacts may be established in an allele-specific manner, we describe an upto-date allele-specific 4C-seq protocol, starting from the selection of allele-specific viewpoints to Illumina sequencing. This protocol has mainly been optimized for cultured mammalian cells, but can be adapted for other cell types with relatively minor changes in initial steps

The Drosophila Fab-7 boundary modulates Abd-B gene activity by guiding an inversion of collinear chromatin organization and alternate promoter use

Summary: Hox genes encode transcription factors that specify segmental identities along the anteroposterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, a feature known as collinearity. In Drosophila, the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive changes in histone modifications, chromatin architecture, and use of boundary elements and cis-regulatory regions. To dissect functional hierarchies, we compare chromatin organization in cell lines and larvae, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary for insulation between 3D domains carrying different histone modifications. Interestingly, we detect a non-canonical inversion of collinear chromatin dynamics at Abd-B, with the domain of active histone modifications progressively decreasing in size. This dynamic chromatin organization differentially activates the alternative promoters of the Abd-B gene, thereby expanding the possibilities for fine-tuning of transcriptional output

The Drosophila Fab-7 boundary element modulates Abd-B gene activity in the genital disc by guiding an inversion of collinear chromatin organization and alternative promoter use

Hox genes encode transcription factors that specify segmental identities along the Antero-Posterior body axis. These genes are organized in clusters, where their order corresponds to their activity along the body axis, an evolutionary conserved feature known as collinearity. In Drosophila , the BX-C cluster contains the three most posterior Hox genes, where their collinear activation incorporates progressive replacement of histone modifications, reorganization of 3D chromatin architecture and sequential activation of boundary elements and cis -regulatory regions. To dissect functional hierarchies, we compared chromatin organization in larvae and in cell lines, with a focus on the Abd-B gene. Our work establishes the importance of the Fab-7 boundary element for insulation between 3D domains marked by different histone modifications. Interestingly, we detected a non-canonical inversion of collinear chromatin dynamics at the Abd-B gene, with the active histone domain decreasing in size. This chromatin organization differentially instructed alternative Abd-B promoter use, thereby expanding the possibilities to regulate transcriptional output

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation

International audienceThe imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian−/− mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5′-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders

Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy

In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5′ end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to gene-rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation

Compostional skew and derivative of the replication timing profile correlate.

<p>Pearson correlation (R values) between the skew and , from different cell lines (Material and Methods). and were calculated in non-overlapping 100 kb windows genome wide (GW) and in the 663 skews N-domains (Ndom). Each 100 kb window was classed as intergenic , genic or genic by majority rule. All p-values are .</p