DNA methylation-histone modification relationships across the desmin locus in human primary cells

Abstract

Background: We present here an extensive epigenetic analysis of a 500 kb region, which encompasses the human desmin gene (DES) and its 5' locus control region (LCR), the only muscle-specific transcriptional regulatory element of this type described to date. These data complement and extend Encyclopaedia of DNA Elements (ENCODE) studies on region ENr133. We analysed histone modifications and underlying DNA methylation patterns in physiologically relevant DES expressing (myoblast/myotube) and non-expressing (peripheral blood mononuclear) primary human cells.

Results: We found that in expressing myoblast/myotube but not peripheral blood mononuclear cell (PBMC) cultures, histone H4 acetylation displays a broadly distributed enrichment across a gene rich 200 kb region whereas H3 acetylation localizes at the transcriptional start site (TSS) of genes. We show that the DES LCR and TSS of DES are enriched with hyperacetylated domains of acetylated histone H3, with H3 lysine 4 di- and tri-methylation (H3K4me2 and me3) exhibiting a different distribution pattern across this locus. The CpG island that extends into the first intron of DES is methylation-free regardless of the gene's expression status and in non-expressing PBMCs is marked with histone H3 lysine 27 tri-methylation (H3K27me3).

Conclusion: Overall, our results constitute the first study correlating patterns of histone modifications and underlying DNA methylation of a muscle-specific LCR and its associated downstream gene region whilst additionally placing this within a much broader genomic context. Our results clearly show that there are distinct patterns of histone H3 and H4 acetylation and H3 methylation at the DES LCR, promoter and intragenic region. In addition, the presence of H3K27me3 at the DES methylation-free CpG only in non-expressing PBMCs may serve to silence this gene in non-muscle tissues. Generally, our work demonstrates the importance of using multiple, physiologically relevant tissue types that represent different expressing/non-expressing states when investigating epigenetic marks and that underlying DNA methylation status should be correlated with histone modification patterns when studying chromatin structure.

Figure 1

Microarray analysis for histone H3 and H4 acetylation across the DES LCR and ENCODE region ENr331 covering 500 kb of human chromosome 2q35. Chromatin (mono-tetranucleosomes) prepared using the nChIP approach was hybridized to a microarray tile, which covers a 500 kb region of chromosome 2q35 and encompasses the DES LCR and DES. Annotated genes taken from the UCSC genome browser are shown as black rectangles in each panel. (A) A peak of histone H3 acetylation can be observed at the beginning of the genes DES, SPEG and ACCN4. Note the grey bar indicating the position of APEG1. (B) Histone H4 acetylation appears over the entire gene rich region and is highly enriched across the DES promoter and LCR in muscle cells. The solitary peak of H4 acetylation downstream of the gene rich region (Panel F) was caused by an excess of Cy3 dye background staining on the corresponding part of the slide and therefore does not represent a true enrichment. Fold enrichment was obtained from an average ratio of two independent experiments.

Figure 2

Fine resolution mapping of histone modifications across the DES LCR and DES in myoblasts and myotubes compared to PBMCs. Chromatin was prepared from myoblasts, myotubes and PBMCs for nChIP (as Figure 1) and amplified using Q-PCR to gain a comprehensive picture of histone modifications status across the DES LCR and DES. (A) Histone H3 acetylation in unfused myoblasts (UF, black bars), in fused myotubes, which express DES 2-fold higher than myoblasts (F, white bars) and in non-expressing PBMCs (grey bars) across DES and the DES LCR. (B) As panel (A) but showing the distribution of histone H4 acetylation. (C) Histone H3K4 di-methylation (H3K4me2) in unfused myoblasts (UF, black bars), in fused myotubes (F) and in non-expressing PBMCs (grey bars) across DES and the DES LCR. (D) As panel (C) but showing histone H3K4me3 enrichment across DES and the DES LCR. Positive controls include the housekeeping genes DNPEP and HTATIP2 and as a non-expressing negative control, HBB. (E) Illustration of DES and DES LCR genomic region. The black rectangles and horizontal arrow denote the exons and direction of transcription of DES respectively. Vertical arrows indicate the relative positions of the DES LCR HS and evolutionarily conserved regions. Vertical bars designated A-J denote the positions of the PCR amplicons following nChIP. Results are presented as fold-enrichment of IP samples over input DNA. Error bars represent the standard deviation about the mean fold-enrichment from at least 2 separate PCRs performed in duplicate from 2 independent nChIP experiments. Enrichment across the muscle-specific DES locus was above non-specific binding background controls (no antibody and IgG) in the myoblast/myotube cultures but not PBMCs. P value < 0.05 denoted by an asterisk (*).

Figure 3

High-resolution DNA methylation mapping of individual CpG dinucleotides within highly conserved regions of the DES LCR. Sodium bisulphite conversion of HindIII-digested human unfused myoblast (UF), fused myotube (F) and PBMC DNA followed by PCR amplification, cloning and sequencing was used to obtain a high-resolution map of the methylation status across GC-rich regions of high sequence homology between mouse, rat and human within the DES LCR. (A) Top line: illustration of the DES and DES LCR HS 1–5 and positions of the evolutionarily conserved elements (vertical arrows). Middle line: CpG density map. Each vertical line represents a single CpG dinucleotide residue. Bottom line: position of PCR amplicons following genomic DNA bisulphate conversion and nChIP (Figure 2). (B) Methylation status of individual CpG sites. Each line represents the result within independent clones of PCR amplified regions. Open/white circles indicate an unmethylated CpG site; closed/black circles, methylated CpG sites. At certain sites circles are missing, sequence data was not available for these individual CpG sites. Amplicons used for nChIP analysis are shown (A-G) by a horizontal double-ended arrow. Positions of sequenced regions relative to the TSS of DES are indicated in kb. Regions of homology within the LCR are marked by a horizontal grey line.

Figure 4

Histone H3 lysine (K) 27 tri-methylation (me3) is highly enriched at the DES CpG island in non-expressing PBMCs. nChIP experiments (Figure 2) were performed using antibodies against H3K27me3. (A) H3K27me3 of DES and the DES LCR in non-expressing PBMCs (grey bars). Non-specific binding was assessed by employing IgG (dark grey bars) and no antibody (stripped bars) samples as controls. Results are presented as fold-enrichment of IP samples over input DNA. Error bars represent the standard deviation about the mean fold-enrichment from at least 2 separate PCRs performed in duplicate from 2 independent nChIP experiments. (B) Illustration of DES and the DES LCR. Vertical arrows indicate the regions of high sequence conservation within each HS site of the LCR and the DES transcriptional start site (TSS) is represented by a horizontal arrow. Black rectangles denote the exons of DES. PCR amplicons (A-J) are shown by vertical black bars. Significant H3K27me3 enrichment was seen only at the DES promoter/CpG island.