Establishing tissue-specific chromatin organization during development of the epidermis. Nuclear architecture of different layers of murine epidermis and the role of p63 and Satb1 in establishing tissue-specific organization of the epidermal differentiation complex locus.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes

The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome.

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi

p63 regulates Satb1 to control tissue-specific chromatin remodeling during development of the epidermis

Genome organizer Satb1 is regulated by p63 and contributes to epidermal morphogenesis by remodeling chromatin structure and gene expression at the epidermal differentiation complex locus

5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells

YesMammalian genomes contain several dozens of large (>0.5 Mbp) lineage-specific gene loci harbouring functionally related genes. However, spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs) in these loci remain poorly understood. TADs are principle units of the genome folding and represents the DNA regions within which DNA interacts more frequently and less frequently across the TAD boundary. Here, we used Chromatin Conformation Capture Carbon Copy (5C) technology to characterize spatial chromatin interaction network in the 3.1 Mb Epidermal Differentiation Complex (EDC) locus harbouring 61 functionally related genes that show lineage-specific activation during terminal keratinocyte differentiation in the epidermis. 5C data validated by 3D-FISH demonstrate that the EDC locus is organized into several TADs showing distinct lineage-specific chromatin interaction networks based on their transcription activity and the gene-rich or gene-poor status. Correlation of the 5C results with genome-wide studies for enhancer-specific histone modifications (H3K4me1 and H3K27ac) revealed that the majority of spatial chromatin interactions that involves the gene-rich TADs at the EDC locus in keratinocytes include both intra- and inter-TAD interaction networks, connecting gene promoters and enhancers. Compared to thymocytes in which the EDC locus is mostly transcriptionally inactive, these interactions were found to be keratinocyte-specific. In keratinocytes, the promoter-enhancer anchoring regions in the gene-rich transcriptionally active TADs are enriched for the binding of chromatin architectural proteins CTCF, Rad21 and chromatin remodeler Brg1. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1 in keratinocytes. Thus, spatial interactions between gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are cell type-specific and involve extensive contacts within TADs as well as between different gene-rich TADs, forming the framework for lineage-specific transcription.This study was supported by the grants 5R01AR064580 and 1RO1AR071727 to VAB, TKS and AAS, as well as by the grants from MRC (MR/ M010015/1) and BBSRC (BB/K010050/1) to VAB

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells

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Cigarette smoke-induced transgenerational alterations in genome stability in cord blood of human F1 offspring

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of gammaH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; gammaH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen

Chromatin architectural proteins CTCF, Rad21 and ATP-dependent chromatin remodeller Brg1 are enriched in the regions involved in the promoter-enhancer spatial interactions at the EDC locus.

<p><b>(a)</b> Schematic map of the EDC containing locus and genome browser view of the normalized ChIP-seq signals for the indicated proteins. The TAD border midpoints are indicated by the green vertical lines. <b>(b)</b> Number of the called ChIP-Seq peaks for the indicated proteins in the individual TADs <b>(c)</b> Percentage of all “true” 5C looping interactions anchoring the regions bound by the indicated proteins. <b>(d)</b> Results of the enrichment analysis of 5C interactions anchoring the regions bound by the indicated proteins in comparison to all the background interactions at the EDC containing locus.–log10 of the p-values are shown (exact Fisher test). <b>(e)</b> Percentage of the significant 5C interactions anchored to the regions bound by the proteins indicated in the left column that are also anchored to the regions bound by the proteins indicated in the top row. <b>(f)</b> Percentage of 5C interaction involving gene enhancers that are anchor the regions bound by the indicated proteins.<b>(g)</b> Results of the enrichment analysis for the 5C interactions involving gene enhancers that are anchored to the regions bound by the indicated proteins in comparison to all background interactions in the EDC locus.–log10 of the p-values are shown (exact Fisher test).</p

Spatial interaction networks between gene enhancers and promoters at the EDC locus in keratinocytes.

<p><b>(a)</b> 5C looping interactions between gene enhancers (top line) and promoters (bottom line) (in red) and gene enhancers with regions not containing gene promoters (bottom line) (in bleu), TAD border midpoints are indicated by the vertical green lines. Schematic organization of the EDC locus is also shown. <b>(b)</b> 5C looping interactions involving <i>Ivl</i> and <i>S100a11</i> gene promoters (bottom line) with their enhancers (top line), TAD border midpoints are indicated by the vertical green lines. <b>(c)</b> 5C looping interactions between the enhancer cluster E2/E3 (top line) and its putative target gene promoters (bottom line), TAD border midpoints are indicated by the vertical green lines. <b>(d)</b> 5C looping interactions between the enhancer E9 (top line) and its putative target gene promoters. <b>(e)</b> Scaling plot showing the normalized average counts versus genomic distances for the 5C looping interaction between gene promoters and enhancers within the TADs (red), and between the TADs (blue).</p

TAD organization of the large (>0.5 Mb) lineage-specific co-regulated gene loci in the mouse genome.

<p><b>(a)</b> Number of TADs occupied by the lineage-specific co-regulated gene loci in the mouse genome in the Embryonic Stem Cells (ESCs) depending of the locus size (based on the Hi-C data published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006966#pgen.1006966.ref009" target="_blank">9</a>]). <b>(b)</b> Genome positions of the lineage-specific co-regulated gene loci in mouse genome and the position of the TAD borders occupied by these loci in the ESCs (mouse genome assembly mmu9), based on the Hi-C data published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006966#pgen.1006966.ref009" target="_blank">9</a>]).</p