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

Abstract

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.

Figure 1. Cfp1 is enriched in non-methylated CpG island chromatin

a, Western blot analysis of non-methylated CGI and bulk chromatin released from purified nuclei using antibodies against selected histone modifications and CpG-binding proteins (normalized to histone H3 levels). An antibody against histone H3 served as a loading control. Modifications associated with transcriptional activity, including H3 acetylated on amino acids K9 and K14, H3K4me3 and H3K4me2 were enriched in CGI chromatin, whereas the elongation and silencing marks H3K36me3, H3K9me3, H3K27me3 and H4K20me3 were depleted. The CXXC domain proteins Cfp1 and Kdm2a also showed enrichment within the CGI fraction. b, Cfp1 ChIP assayed by qPCR across the X-linked mouse Xist locus. Vertical strokes beneath the plot represent CpGs within the locus and the black bar above demarcates the CGI. The open box below the CpG map shows the region amplified for bisulphite analysis. IP, immunoprecipitation. c, Bisulphite analysis of input chromatin (female brain) and chromatin immunoprecipitated with Cfp1 antibodies and control MeCP2 antibodies. Twelve representative clones are shown from the total number sequenced (number in brackets). Solid and open circles represent methylated and non-methylated CpGs, respectively. Uncharacterized CpGs are represented as gaps.

Figure 2. Genome-wide ChIP sequencing shows a tight association between Cfp1 and H3K4me3 at CGIs

a, Typical Cfp1 ChIP-Seq profiles from whole mouse brain. For comparison, we also carried out H3K4me3 ChIP-Seq. The data were aligned with non-methylated CGIs mapped in mouse brain using a CXXC affinity column. The panel shows a typical region of the genome from chromosome 4 (nucleotides 126,333,759–127,054,849) demonstrating the coincidence of Cfp1 and H3K4me3 peaks with CGIs. A subset of genes is labelled (RefSeq). Two CGIs that lack H3K4me3 and Cfp1 coincide with sites of H3K27me3 binding (red rectangles; data of ref. 30 for mouse brain). b, Venn diagram showing strong overlap between H3K4me3 and Cfp1 peaks in mouse brain chromatin, but minimal overlap with H3K27me3.

Figure 3. Depletion of Cfp1 results in reduced H3K4 trimethylation levels at CpG islands

a, Expression of three short hairpin RNAs in NIH3T3 fibroblasts reduced Cfp1 messenger RNA levels to 15% compared with vector-only transfected control cells. Expression of Cfp1 relative to Gapdh in control cells is set to 1. The inset shows reduction of Cfp1 by western blotting. Error bars indicate s.d. (n = 3) b, c, Gross morphology (b) and growth rate (c) of Cfp1-depleted versus vector-only transformant cells. Cells were plated at low density and monitored at the times shown using a haemocytometer. Original magnification, ×200. d, ChIP qPCR using Cfp1, H3K4me3 and H3K27me3 antibodies at selected loci in vector-only control and Cfp1-depleted NIH3T3 cells. The results were replicated with an independent clone expressing the same shRNA combination (data not shown) and with each of two individual shRNA constructs (see Supplementary Fig. 3).

Figure 4. Artificial promoterless CpG-rich sequences recruit Cfp1 and generate new H3K4me3 peaks in mouse ES cells

a, The TCβ44 ES cell line carries adjacent promoterless eGFP and bacterial puromycin-resistance sequences (black bars) inserted together within the 3′ untranslated region of the Nanog gene. The positions of CGIs and H3K4me3 peaks at this locus in wild-type ES cells are shown below the map. DNA methylation within the insertion was determined by bisulphite sequencing of 306-bp (eGFP) and 275-bp (Puro) segments of the inserted sequence. b, ChIP qPCR across the region containing the insertion using antibodies against Cfp1, RNA polymerase II (Pol2) and H3K4me3. The dotted line plots CpG density in a 500-bp window with a 100-bp step size. Vertical strokes below the graph mark CpG sites within and surrounding the insertion. c, A second ES cell line carried an eGFP coding sequence (black bar) inserted into the 3′ untranslated region of the X-linked Mecp2 gene. CGIs and H3K4me3 peaks at this locus in wild-type ES cells are shown below. d, ChIP qPCR across the region containing the insertion (black bar) using antibodies against Cfp1, RNA polymerase II and H3K4me3. e, Bisulphite sequence analysis determined the methylation status of input and DNA immunoprecipitated by the Cfp1 and H3K4me3 antibodies. The percentage of non-methylated CpGs is shown below each panel. H3K4me3 ChIP data in b and d used different commercial antibodies with differing affinities (see Supplementary Table 2).