Tissue-Specific Epigenetics of Atherosclerosis-Related ANGPT and ANGPTL Genes

Figure 1.. Expression of ANGPT and ANGPTL genes in different tissues. RNA-seq data are from GTEx [32] for ten of the available tissue types and are given as median TPM for ANGPT1 and ANGPT2 (A) and five of the ANGPTL genes (B). Note difference scales of the y-axis in (A) and (B). For quantitative data from more tissue types and related genes, see Supplementary Table 1. Adipose-visc: Adipose from visceral tissue; Skel muscle: Skeletal muscle; TPM: Transcripts per million.

Figure 2.. ANGPTL3 displays liver-specific enhancer and promoter chromatin and DNA hypomethylation that correlates with its liver-specific expression. (A) ANGPTL3 resides in an intron of DOCK7, a guanine nucleotide exchange factor-encoding gene (chr1:63,047,768–63,160,178). Blue, RefSeq gene isoforms; dark red, an additional Ensembl transcript-encoding isoform. (B) The color code indicates predicted chromatin states [28]: strong, weak, or bivalent promoter or enhancer; transcribed; repressed (H3K27me3 strong or weak, H3K9me3 or H3K9me3 plus H3K36me3); or low-signal chromatin. (C) TF binding in HepG2 cells as determined by TF ChIP-seq is indicated by the given color code. (D) CpG islands [30] and bisulfite-seq tracks, which are plotted as average % methylation at CpGs; blue horizontal bars, regions that display statistically significant hypomethylation relative to the rest of the same genome [36]. The dotted boxes indicate the large region exhibiting liver-specific DNA hypomethylation in (D). Black dots indicate additional regions of hypomethylation overlapping enhancer chromatin. (E) RNA-seq data from ENCODE for individual samples [29,30] with the vertical viewing range set at 0–10 instead…

Figure 3.. ANGPTL8 exhibits intragenic enhancer chromatin overlapping DNA hypomethylation specifically in liver, which has the highest expression. (A) ANGPTL8 is embedded in intron 14 of 47 introns of DOCK6 (chr19:11,339,832–11,363,081). (B–D) Predicted chromatin states, TF binding sites and DNA methylation profile from bisulfite-seq as in Figure 2. Dotted boxes in (B) and (C) indicate regions where liver-associated DNA hypomethylation was seen. (E) DNA methylation profiling by RRBS (with an 11-color gradient) including cell cultures for which bisulfite-seq DNA are not available. (F) and (G), RNA seq data as in Figure 2. MCF-7 and LNCaP, breast and prostate cancer cell lines, respectively; blue highlighting in panel (C), two neighboring RXRA sites and a HNF4A site that overlaps one of them. ESC: Embryonic stem cell; Expr: Expression; fib: Fibroblast; HepG2: Hepatocarcinoma cell; HMEC: Human mammary epithelial cell; HUVEC: Human umbilical vein endothelial cell; K562: Myelogenous leukemia cells; LCL: Lymphoblastoid cell line; Myob: Myoblast; PBMC: Peripheral blood mononuclear cells; RRBS: Reduced representation bisulfite sequencing; Sk muscle: Skeletal muscle; TF: Transcription factor.

Figure 4.. ANGPT2 displays atherosclerosis-associated changes in DNA methylation in aorta. (A) ANGPT2 resides in an intron of long isoforms of Microcephalin 1, MCPH1 (chr8:6,258,632-6,509,865). (B) Chromatin state segmentation as in Figure 2. (C) Statistically significant DMRs (hypermethylated in red or hypomethylated in blue) in an atherosclerotic aorta sample versus control aorta from the same individual or vs three control aorta samples as determined from analysis of bisulfite-seq profiles. (D) CpG islands and bisulfite-seq profiles; green arrow, exon-1 subregion previously found to be hypomethylated in cancers [51]. (E) and (F), RNA-seq profiles as described for Figure 2. (G) Zoomed-in view of chromatin segmentation tracks from panel (B) showing just ANGPT2 and most of the aorta-specific enhancer regions around it (chr8 : 6, 340, 035–6, 456, 867). Dotted boxes, regions of aorta-specific DNA hypomethylation and enhancer chromatin; green arrows, previously studied exon 1 region described in the text. DMR: Differentially methylated region; fib: Fibroblast; HepG2: Hepatocarcinoma cell; HUVEC: Human umbilical vein endothelial cell; LCL: Lymphoblastoid cell line; Myob: Myoblast; PBMC:…

Figure 5.. The gene encoding miR-145 is embedded in an aorta/ovary/heart/osteoblast/lung fibroblast super-enhancer that overlays atherosclerosis-associated hypermethylated regions. (A) Two RefSeq isoforms (CARMN) that overlap the miR-145- and miR-143-encoding sequences and six of the ten Ensembl isoforms in this region, including the only Ensembl isoform that overlaps miR-143-encoding DNA (chr5 : 148, 781, 015–148, 839, 604). (B) Chromatin state segmentation as in Figure 2. (C) Atherosclerosis-associated DMRs as in Figure 4. (D) Bisulfite-seq as in Figure 2. (E) Short RNA-seq (<0.20 kb); the position of the sequences encoding the pre-miRNAs is indicated. (F), RNA-seq (‘Long’ RNA-seq for RNAs >0.2 kb) as in Figure 2 except that the vertical viewing range was 0–300 instead of 0–30 as in other figures. (G) CAGE (TSS from Hidden Markov Modeling for pooled replicates; [29]); green arrow, the position of the putative 1.4-kb MIR145 promoter region mentioned in the text. (H) Median RPKM values from RNA-seq for tissues. The CpG islands are not shown because there were none in this region that fit the definition used by the UCSC Genome Browser [30]. CAGE: 5′ cap analysis of gene expression; DMR:…

Figure 6.. A cartoon summarizing correlations of tissue-specific DNA hypomethylation, active promoter or enhancer chromatin with tissue-specific expression of the studied ANGPT or ANGPTL genes. 2, L1, L2, L3, L4 and L8 denote ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. Assoc: Associated.