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

Mechanisms and consequences of DNA damage, response and apoptosis in spermatozoa

DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-IIβ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer.EThOS - Electronic Theses Online ServiceEuropean Union's 6th Framework project Newborns and genotoxic exposure risk (NewGeneris), British Council's United Kingdom Indian Education Research Initiative (UKIER)GBUnited Kingdo

Mechanisms and consequences of DNA damage, response and apoptosis in spermatozoa

DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-IIβ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer.EThOS - Electronic Theses Online ServiceEuropean Union's 6th Framework project Newborns and genotoxic exposure risk (NewGeneris), British Council's United Kingdom Indian Education Research Initiative (UKIER)GBUnited Kingdo

Pcf11 orchestrates transcription termination pathways in yeast

In Saccharomyces cerevisiae, short noncoding RNA (ncRNA) generated by RNA polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3, and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore, mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA, and restricted Pol II CTD Ser2 phosphorylation and Sen1–Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar, as both accumulate RNA:DNA hybrids and display Pol II pausing downstream from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo

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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo