Robert S. Illingworth

CpG islands -'a rough guide'

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites termed CpG islands (CGIs). CGIs generally lack DNA methylation and associate with the majority of annotated gene promoters. Many studies, however, have identified examples of CGI methylation in malignant cells, leading to improper gene silencing. CGI methylation also occurs in normal tissues and is known to function in X‐inactivation and genomic imprinting. More recently, differential methylation has been shown between tissues, suggesting a potential role in transcriptional regulation during cell specification. Many of these tissue‐specific methylated CGIs localise to regions distal to promoters, the regulatory function of which remains to be determined.

A novel CpG island set identifies tissue-specific methylation at developmental gene loci.

CpG islands (CGIs) are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%–8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

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. 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. 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. 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.

Neuronal MeCP2 Is Expressed at Near Histone-Octamer Levels and Globally Alters the Chromatin State

MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions, and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner.

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells.

During differentiation, thousands of genes are repositioned toward or away from the nuclear envelope. These movements correlate with changes in transcription and replication timing. Using synthetic (TALE) transcription factors, we found that transcriptional activation of endogenous genes by a viral trans-activator is sufficient to induce gene repositioning toward the nuclear interior in embryonic stem cells. However, gene relocation was also induced by recruitment of an acidic peptide that decondenses chromatin without affecting transcription, indicating that nuclear reorganization is driven by chromatin remodeling rather than transcription. We identified an epigenetic inheritance of chromatin decondensation that maintained central nuclear positioning through mitosis even after the TALE transcription factor was lost. Our results also demonstrate that transcriptional activation, but not chromatin decondensation, is sufficient to change replication timing. The position of a gene in the cell nucleus is dictated by the compaction state of its chromatin wrapper. Unpacking for travel to the nuclear interior The position of a gene within the cell nucleus is correlated with its activity. Those near the nuclear periphery are generally repressed, whereas those in the center are (or will be) active. It is not clear whether this relocalization is a cause or a consequence of gene regulation. Therizols et al. found that transcriptional activation or simply chromatin decondensation both drove the relocation of genes to the interior of the nucleus. The nuclear position was maintained in daughter cells, suggesting that the cell has an epigenetic memory of the genes position within the nucleus. Science, this issue p. 1238

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each others binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B.

Immunostaining of modified histones defines high-level features of the human metaphase epigenome.

BackgroundImmunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies.ResultsBy immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3.ConclusionsAt metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.

PRC1 and PRC2 Are Not Required for Targeting of H2A.Z to Developmental Genes in Embryonic Stem Cells

The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner.

Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome

The possibility that alterations in DNA methylation are mechanistic drivers of development, aging and susceptibility to disease is widely acknowledged, but evidence remains patchy or inconclusive. Of particular interest in this regard is the brain, where it has been reported that DNA methylation impacts on neuronal activity, learning and memory, drug addiction and neurodegeneration. Until recently, however, little was known about the ‘landscape’ of the human brain methylome. Here we assay 1.9 million CpGs in each of 43 brain samples representing different individuals and brain regions. The cerebellum was a consistent outlier compared to all other regions, and showed over 16 000 differentially methylated regions (DMRs). Unexpectedly, the sequence characteristics of hypo- and hypermethylated domains in cerebellum were distinct. In contrast, very few DMRs distinguished regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable in these regions. These results lead to the surprising conclusion that, with the exception of cerebellum, DNA methylation patterns are more homogeneous between different brain regions from the same individual, than they are for a single brain region between different individuals. This finding suggests that DNA sequence composition, not developmental status, is the principal determinant of the human brain DNA methylome.