Most human gene promoters are embedded within CpG islands that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity. Here, a zinc-finger protein Cfp1 is found to be associated with non-methylated CpG islands and sites of H3K4me3 genome-wide in vivo. Cfp1 is part of the Setd1 complex which trimethylates H3K4. Artificial CpG clusters are shown to recruit Cfp1, leading to novel peaks of H3K4me3. Therefore a primary function of non-methylated CpG islands might be to genetically determine the local chromatin modification state. Most human gene promoters are embedded within CpG islands that lack DNA methylation and coincide with sites at which histone H3 lysine 4 is trimethylated (H3K4me3 sites). Here, a zinc-finger protein, Cfp1, is found to be associated with non-methylated CpG islands and H3K4me3 sites throughout the genome in the mouse brain. A primary function of non-methylated CpG islands might be to genetically determine the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins. CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides1,2. 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 activity3,4. 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 vitro5,6. 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.

CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs (>10,000) are “orphans” that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development. In the decade since the sequence of the human genome was announced, efforts have been made to annotate all genes with their regulatory sequences. CpG islands are short regions containing the sequence CG at high density that map to regions controlling the expression of most human genes (known as promoters). Using a biochemical method, we have identified and mapped all CpG islands in the human and mouse genomes and find that over half are remote from known gene promoters—so-called “orphans.” Mice, which were thought to possess far fewer CpG islands than humans, turn out to have a very similar number. Surprisingly, orphan CpG islands in both species often mark hitherto unknown promoters. The activity of these novel promoters is particularly dynamic during normal development, as they are often silenced by DNA methylation. In colorectal cancers, however, aberrant DNA methylation affects all CpG islands equally.

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.

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.