MMDiff: quantitative testing for shape changes in ChIP-Seq data sets

BACKGROUND: Cell-specific gene expression is controlled by epigenetic modifications and transcription factor binding. While genome-wide maps for these protein-DNA interactions have become widely available, quantitative comparison of the resulting ChIP-Seq data sets remains challenging. Current approaches to detect differentially bound or modified regions are mainly borrowed from RNA-Seq data analysis, thus focusing on total counts of fragments mapped to a region, ignoring any information encoded in the shape of the peaks. RESULTS: Here, we present MMDiff, a robust, broadly applicable method for detecting differences between sequence count data sets. Based on quantifying shape changes in signal profiles, it overcomes challenges imposed by the highly structured nature of the data and the paucity of replicates. We first use a simulated data set to compare the performance of MMDiff with results obtained by four alternative methods. We demonstrate that MMDiff excels when peak profiles change between samples. We next use MMDiff to re-analyse a recent data set of the histone modification H3K4me3 elucidating the establishment of this prominent epigenomic marker. Our empirical analysis shows that the method yields reproducible results across experiments, and is able to detect functional important changes in histone modifications. To further explore the broader applicability of MMDiff, we apply it to two ENCODE data sets: one investigating the histone modification H3K27ac and one measuring the genome-wide binding of the transcription factor CTCF. In both cases, MMDiff proves to be complementary to count-based methods. In addition, we can show that MMDiff is capable of directly detecting changes of homotypic binding events at neighbouring binding sites. MMDiff is readily available as a Bioconductor package. CONCLUSIONS: Our results demonstrate that higher order features of ChIP-Seq peaks carry relevant and often complementary information to total counts, and hence are important in assessing differential histone modifications and transcription factor binding. We have developed a new computational method, MMDiff, that is capable of exploring these features and therefore closes an existing gap in the analysis of ChIP-Seq data sets

Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.Spanish Government SAF2010-21017Spanish Government BES-2011-0473

Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of “ectopic” H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents “leakage” of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3

Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA

MBD1, a member of the methyl-CpG-binding domain family of proteins, has been reported to repress transcription of methylated and unmethylated promoters. As some MBD1 isoforms contain two DNA-binding domains—an MBD, which recognizes methylated DNA; and a CXXC3 zinc finger, which binds unmethylated CpG—it is unclear whether these two domains function independently of each other or if they cooperate in facilitating recruitment of MBD1 to particular genomic loci. In this report we investigate DNA-binding specificity of MBD and CXXC3 domains in vitro and in vivo. We find that the methyl-CpG-binding domain of MBD1 binds more efficiently to methylated DNA within a specific sequence context. We identify genes that are targeted by MBD1 in human cells and demonstrate that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to specific sites at these loci, while DNA binding by the CXXC3 motif is largely dispensable. In summary, the binding preferences of MBD1, although dependent upon the presence of methylated DNA, are clearly distinct from those of other methyl-CpG-binding proteins, MBD2 and MeCP2

Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions

CpG islands influence chromatin structure via the CpG-binding protein Cfp1

CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides(1,2). Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity(3,4). In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro(5,6). Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins