Project description:The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation [ChIP-Seq]
Project description:The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type–specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K36me2/K4me1 marks transcribed enhancers, while H3K36me3/K4me1 and H3K79me2/K4me1 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics.
Project description:The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation [RNA-Seq]
Project description:In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription, translocation, and homologous recombination. However, DNA hypomethylation occurs during specific developmental stages (e.g. preimplantation embryos) and in certain cell types (e.g., primordial germ cells). The absence of dysregulated repetitive elements in these cells suggests the existence of alternative mechanisms that prevent genome instability triggered by DNA hypomethylation. In this report, we seek to elucidate the factors that play a critical role in ensuring genome stability by focusing on DAXX and ATRX, two proteins that have been linked to transcriptional control and epigenetic regulation. We carried out ChIP-seq and RNA-seq analyses to compare the genome-wide binding and transcriptome profiles of DAXX and ATRX in mouse ES (mES) cells triple knocked out for the three mammalian DNA methyltransferases (DNMTs) (TKO cells) to those in wildtype mES cells. Our data indicate that DAXX and ATRX are distinct in their chromatin-binding profiles and highly co-enriched at tandem repetitive elements. Global DNA hypomethylation, as was the case in TKO cells, further promoted the recruitment of the DAXX/ATRX complex to tandem repeat sequences including IAP (intracisternal A‐particle) retrotransposons and telomeres. Inhibition of DAXX or ATRX in cells with hypomethylated genomes (e.g., TKO cells, mES cells cultured in ground-state conditions, and preimplantation embryos) increased aberrant transcriptional de-repression of repeat elements and dysfunction at telomeres. Furthermore, we provide evidence that DAXX/ATRX-dependent silencing may occur through DAXX’s interaction with SUV39H1 and increased H3K9me3 on repetitive sequences. Our study suggests that DAXX and ATRX are important for safeguarding the genome, particularly in silencing repetitive elements in the absence of DNA methylation. We tested the hypothesis that the DAXX/ATRX complex participates in protecting repetitive elements in the absence of DNA methylation. To this end, we investigated genome-wide chromatin targeting of DAXX and ATRX in wildtype mES cells, and in mES cells that exhibit extensive loss of DNA methylation due to homozygous knockout of all three DNA.
Project description:Background: Global DNA methylation contributes to genomic integrity by supressing repeat associated transposition events. Several chromatin factors are required in addition to DNA methyltransferases to maintain DNA methylation at intergenic and satellite repeats. Embryos lacking Lsh, a member of the SNF2 superfamily of chromatin helicases, are hypomethylated. The interaction of Lsh with the de novo methyltransferase, Dnmt3b, facilitates the deposition of DNA methylation at stem cell genes. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences. Results: We used HELP-seq to map genome wide DNA methylation patterns in Lsh-/- and Dnmt3b-/- somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh-/- cells: LTR-retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP (Intracisternal A-type particle) LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed inLsh-/- cells. LTR hypomethylation in Dnmt3b-/- cells is moderate and hypomethylated repetitive elements (IAP, LINE-1 and satellite) are silent. Chromatin immunoprecipitation (ChIP) indicates that repressed LINE-1 elements gain H3K4me3, but H3K9me3 levels are unaltered in Lsh-/- cells, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh-/- cells. Conclusions: Our study emphasizes that regulation of repetitive elements by DNA methylation is selective and context dependent. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 in somatic cells to enforce repeat silencing thus contributing to genomic integrity. Two pairs of genomic samples compared: WT and Lsh-/- DNA isolations from tail-tip fibroblasts; WT and Dnmt3b knockout DNA isolations from mouse embryonic fibroblasts.
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Background: Global DNA methylation contributes to genomic integrity by supressing repeat associated transposition events. Several chromatin factors are required in addition to DNA methyltransferases to maintain DNA methylation at intergenic and satellite repeats. Embryos lacking Lsh, a member of the SNF2 superfamily of chromatin helicases, are hypomethylated. The interaction of Lsh with the de novo methyltransferase, Dnmt3b, facilitates the deposition of DNA methylation at stem cell genes. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences. Results: We used HELP-seq to map genome wide DNA methylation patterns in Lsh-/- and Dnmt3b-/- somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh-/- cells: LTR-retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP (Intracisternal A-type particle) LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed inLsh-/- cells. LTR hypomethylation in Dnmt3b-/- cells is moderate and hypomethylated repetitive elements (IAP, LINE-1 and satellite) are silent. Chromatin immunoprecipitation (ChIP) indicates that repressed LINE-1 elements gain H3K4me3, but H3K9me3 levels are unaltered in Lsh-/- cells, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh-/- cells. Conclusions: Our study emphasizes that regulation of repetitive elements by DNA methylation is selective and context dependent. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 in somatic cells to enforce repeat silencing thus contributing to genomic integrity. Two pairs of RNA samples compared: WT and Lsh-/- RNA isolations from tail-tip fibroblasts; WT and Lsh-/- RNA isolations from E13.5 mouse embryos.