Project description:We report the application of Next-generation sequencing (NGS) for high-throughput profiling of N4-acetylcytosine (4acC) DNA modification in Arabidopsis thaliana. We find that 4acC peaks mostly distribute in the euchromatin regions and are present in nearly half of the protein-coding genes in Arabidopsis. 4acC mainly locates around transcription start sites (TSSs) of protein-coding genes and positively correlates with gene expression level . Compared to non-4acC marked genes, 4acC marked genes display a higher methylation level of 5mC in the CG context within coding regions, but a lower methylation level of 5mC in the CHG and CHH contexts. In addition, 4acC peaks showed higher overlaps with active histone modification marks than by chance, but similar overlaps with repressive histone modification marks. We observed complex cooperative interactions among 4acC, 5mC, and histone modifications on gene transcription. 4acC containing genes with low level of mCG, or active histone modification marks displayed a higher expression. In sum, we have uncovered 4acC as a hitherto unknown epigenetic DNA mark that is associated with gene expression in Arabidopsis, and suggest potential cross-talks among epigenetic marks for gene expression. Overall design: Examination of 4acC DNA modification in Arabidopsis thaliana.
Project description:Most genome-level analysis treats the two parental alleles equivalently, yet diploid genomes contain two parental genomes that are often epigenetically distinct. While single nucleotide polymorphisms (SNPs) can be used to distinguish these genomes, it would be useful to develop a generalized strategy for identifying candidate genes or regions showing allele-specific differences, independent of SNPs. We have explored this problem by looking for overlapping marks in the genome related to both euchromatin (histone H3 dimethyl lysine-4 [H3K4Me2]) and heterochromatin (DNA methylation [DNAm]). "Double hits" were defined by the intersection of H3K4Me2 and DNAm. For the top 5% of marks, defined by a sliding window, imprinted gene regions were enriched for double hits 5.4-fold. When the location information of CTCF binding sites were integrated, the "triple hits" were enriched 76-fold for known imprinted genes in the regions studied. The double hits in imprinted genes were found to occur usually at the site of alternative or antisense transcripts. In addition, four of four imprinted genes tested showing double hits also showed allele-specific methylation. We suggest that overlapping euchromatin/heterochromatin marks are common and are enriched for epigenetically distinct parental chromosome regions. Furthermore, we developed a novel approach to identifying allele-specific marks that is SNP independent, by fractionating using H3K4Me2 antibodies followed by DNA methylation analysis.
Project description:Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin.
Project description:The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure with activating and repressive histone modifications that promote latent gene expression but suppress lytic gene expression. Here, we report a comprehensive epigenetic study of the recruitment of chromatin regulatory factors onto the KSHV genome during the pre-latency phase of KSHV infection. This demonstrates that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and accompanied by the transient induction of a limited number of lytic genes. Interestingly, temporary expression of the RTA protein facilitated the increase of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both Polycomb Repressive Complex 1 and 2. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally-ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection versus lytic replication of KSHV.
Project description:In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.
Project description:Human chromosomal regions enriched in segmental duplications are subject to extensive genomic reorganization. Such regions are particularly informative for illuminating the evolutionary history of a given chromosome. We have analyzed 866 kb of Y-chromosomal non-palindromic segmental duplications delineating four euchromatin/heterochromatin transition regions (Yp11.2/Yp11.1, Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2). Several computational methods were applied to decipher the segmental duplication architecture and identify the ancestral origin of the 41 different duplicons. Combining computational and comparative FISH analysis, we reconstruct the evolutionary history of these regions. Our analysis indicates a continuous process of transposition of duplicated sequences onto the evolving higher primate Y chromosome, providing unique insights into the development of species-specific Y-chromosomal and autosomal duplicons. Phylogenetic sequence comparisons show that duplicons of the human Yp11.2/Yp11.1 region were already present in the macaque-human ancestor as multiple paralogs located predominantly in subtelomeric regions. In contrast, duplicons from the Yq11.1/Yq11.21, Yq11.23/Yq12, and Yq12/PAR2 regions show no evidence of duplication in rhesus macaque, but map to the pericentromeric regions in chimpanzee and human. This suggests an evolutionary shift in the direction of duplicative transposition events from subtelomeric in Old World monkeys to pericentromeric in the human/ape lineage. Extensive chromosomal relocation of autosomal-duplicated sequences from euchromatin/heterochromatin transition regions to interstitial regions as demonstrated on the pygmy chimpanzee Y chromosome support a model in which substantial reorganization and amplification of duplicated sequences may contribute to speciation.
Project description:N4-acetylcytidine (ac4C) is a highly conserved RNA modification and is the first acetylation event described in mRNA. ac4C in mRNA has been demonstrated to be involved in the regulation of mRNA stability, processing and translation, but the exact means by which ac4C works remain unclear. In addition, ac4C is widely distributed within the human transcriptome at physiologically relevant levels and so far only a small fraction of modified sequences have been detected by experiments. In this study, we developed a predictor of ac4C sites in human mRNA named PACES to help mining possible modified motifs. PACES combines two random forest classifiers, position-specific dinucleotide sequence profile and K-nucleotide frequencies. With genomic sequences as input, PACES gives possible modified sequences based on the training model. PACES is freely available at http://www.rnanut.net/paces/.
Project description:N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome. 4mC-TAB-seq
Project description:Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.
Project description:A characteristic spatial distribution of the main chromatin fractions is observed in most mammalian cell nuclei, with euchromatin localized in the interior and heterochromatin at the nuclear periphery. It has been shown that interactions of heterochromatin with the nuclear lamina are necessary to establish this conventional architecture. Adipocytes are specific cells in which a reduction in lamin A/C expression is observed. We hypothesize that the loss of lamin A/C during adipogenic differentiation of mesenchymal stem cells (MSCs) may be associated with the reorganization of the main classes of chromatin in the nucleus. Thus, in this study, we examine the abundance and nuclear distribution of selected heterochromatin (H3K9me3, H3K27me3 and H4K20me3) and euchromatin (H4K8ac, H3K4me3 and H3K9ac) histone marks during in vitro adipogenesis, using the pig as a model organism. We found that not only did the expression of lamin A/C decrease in our differentiation system, but so did the expression of lamin B receptor (LBR). The level of two heterochromatin marks, H3K27me3 and H4K20me3, increased during differentiation, while no changes were observed for H3K9me3. The levels of two euchromatin histone marks, H4K8ac and H3K9ac, were significantly higher in adipocytes than in undifferentiated cells, while the level of H3K4me3 did not change significantly. The spatial distribution of all the examined histone marks altered during in vitro adipogenesis. H3K27me3 and H4K20me3 moved towards the nuclear periphery and H3K9me3 localized preferentially in the intermediate part of adipocyte nuclei. The euchromatin marks H3K9ac and H3K4me3 preferentially occupied the peripheral part of the adipocyte nuclei, while H4K8ac was more evenly distributed in the nuclei of undifferentiated and differentiated cells. Analysis of the nuclear distribution of repetitive sequences has shown their clustering and relocalization toward nuclear periphery during differentiation. Our study shows that dynamic changes in the abundance and nuclear distribution of active and repressive histone marks take place during adipocyte differentiation. Nuclear reorganization of heterochromatin histone marks may allow the maintenance of the nuclear morphology of the adipocytes, in which reduced expression of lamin A/C and LBR is observed.