Landes Bioscience www.landesbioscience.com Supplemental Material to: Koji Tsumagari, Carl Baribault, Jolyon Terragni, Katherine E. Varley, Jason Gertz, Sirharsa Pradhan, Melody Badoo, Charlene M. Crain, Lingyun Song, Gregory E. Crawford, Richard M. Myers, Michelle Lacey and Melanie Ehrlich Early de novo DNA methylation and prolonged demethylation in the muscle lineage Epigenetics 2013; 8(3) http://dx.doi.org/10.4161/epi.23989 http://www.landesbioscience.com/journals/epigenetics/article/23989 Supplementary Material for “Early de novo DNA methylation and prolonged demethylation in the muscle lineage” Supplementary Figures Figure S1. Myogenic hypermethylated CpGs in homeobox genes were clustered much closer to the TSS in skeletal muscle than in myogenic precursor cells. The distances between the transcription start site (TSS) and myogenic hypermethylated sites in skeletal muscle or myoblasts plus myotubes (MbMt) were determined with the GREAT tool for analysis of cis regulatory elements. 1 (a) MbMt hypermethylated sites (MbMt vs. non-muscle cell cultures) associated with homeobox genes were moderately enriched in the 100-kb region centered on the TSS when compared with total MbMt-hypermethylated sites. (b) Muscle hypermethylated sites (skeletal muscle vs. nonmuscle tissues) associated with homeobox genes showed strong enrichment very close to the TSS when compared with total muscle-hypermethylated sites. This enrichment of hypermethylated sites was seen in the 10-kb region centered on the TSS in skeletal muscle rather than more broadly in the 100-kb region centered on the TSS as for Mb and Mt. a Myoblasts & myotubes b Muscle All MbMt hyper meth sites All muscle hypermeth sites MbMt hyper meth sites for homeobox genes Muscle hypermeth sites for homeobox genes Distance of MbMt hypermeth sites to TSS Distance of muscle hypermeth sites to TSS 1 Figure S2a. PAX3 and CCDC140: Myogenic DNA hypermethylation and preferential H3K9 trimethylation was seen not only in PAX3, but also in and near the overlapping CCDC140 gene. (a) PAX3 and CCDC140, which are divergently transcribed, are weakly expressed in Mb and skin fibroblasts. The following profiles are shown (http://genome.ucsc.edu) for the PAX3 and CCDC140 region (chr2:223,058,677- 223,174,523; all coordinates for hg19): the mouse net track from the UCSC Genome Browser, which shows sequence conservation between the mouse and human genomes; Chromatin State Segmentation analysis (color code designations are shown below with the addition that blue indicates a predicted insulator; ENCODE/Broad); histone H3K9me3 enrichment by ChIP-seq (mean signal value across the interval; ENCODE/Broad); statistically significant myogenic hypo- or hypermethylated sites (p <0.01; this study); and Long RNA-seq tracks for strand-specific RNA-seq (>200 nt poly(A)+ RNA; vertical viewing range, 1 - 20; ENCODE/Cold Spring Harbor). Skin fibroblast 3 (Fib3) and Mb were the only cell types of eight examined (LCL, NHEK, NHLF, HUVEC, ESC, and HMEC; Table S5 and data not shown) displaying transcripts from these genes in RNA-seq. These genes were embedded in chromatin with significantly elevated H3K9Me3 signal in Mb and Mt, but not in fibroblasts. 2 Figure S2b. 6.7 kb downstream of CCDC140, melanocytes have specific CpG methylation in a region that partly overlaps CpG sites with elevated DNA methylation in control Mb and Mt. A box denotes the distinctively high DNA methylation in melanocytes throughout this region. Melanocytes strongly express and Mb weakly express CCDC140 and the adjacent PAX3 (Table S5). Only the 5’ subregion also had considerable methylation in control Mb and Mt. In the 3’ subregion, Mb and Mt displayed a DNaseI hypersensitive site (DHS) missing in melanocytes. 5’ to this DHS, Mb and Mt were enriched in H3K9me3. SGPP2, the gene immediately distal to and 126 kb downstream of CCDC140, is not preferentially expressed in myogenic cells. The region shown in this figure is located ~2 kb downstream of the end of Figure S2a. 3 Figure S3. TBX1, which is highly expressed in myogenic cells, had 131 MbMt-hypermethylated sites in the promoter region and the gene body, which probably help positively control expression of the gene as 5hmC residues. (a) Mb and HUVEC, which selectively express this gene, are highly methylated in the body of the gene, and Mb are also hypermethylated in the 5’ gene region and have a hypomethylated site 2.6 kb upstream of the TSS. The epigenetic profiles for TBX1 (chr22:19,732,116-19,771,300) are shown as above except that the vertical viewing range was 1 - 100 for strand-specific RNA. At the gene-level resolution, it is difficult to visualize many of the myogenic DM sites in the RRBS data tracks. The left insert shows an enlarged view of the myogenic DNA hypomethylation at - 2.6 kb (chr22:19,741,617-19,741,711) with the following nonmuscle cell types: Fib1, Fib2, osteoblasts, HRCEpiC, HRE, LCL, HMEC, astrocytes, HCPEpiC (choroid plexus epithelial cells, hypomethylated like Mb and Mt), HIPEPiC, HRPEpiC, NHBE, and ESC. The hypomethylation of this TBX1-upstream site in choroid plexus cells suggests that this gene, which has been linked to neurological function,2 has a special role in choroid plexus-related neurological activity.3 4 Figure S3 (b). TBX1 had a large cluster of MbMt- hypermethylated CpG sites that overlaps a gap in the H3K36Me3 signal. This cluster (yellow highlighting) also overlaps the position of one of two TBX1 CTCF sites (orange boxes; ENCODE/Broad) seen in various other cell types but not in Mb and Mt. A second large cluster of MbMt- hypermethylated sites (pink highlighting) borders an intragenic region with promoter- like H3K4 di- and trimethylation in Mb, Mt, and HUVEC. A 3’ region exhibited enhancer-type modifications specific to myogenic cells (brown box). RNA-seq data (Table S5) indicate that the predominant transcript in Mb corresponds to the second gene isoform shown below. The high level of expression of this gene combined with the enrichment in hypermethylated sites both in the extended promoter region and in the gene body in subregions with little or no H3K36me3 or HK27Me3 make it likely that the myogenic DM sites throughout the gene region are hydroxymethylated and not methylated C residues. 5 Figure S4. OBSCN: Muscle-specific DNA hypomethylation at 73 sites and MbMt-specific DNA hypermethylation at two sites. OBSCN’s epigenetic marks and RNA-seq data (chr1:228,378,622-228,575,138) are shown as in previous figures but the RefSeq Gene track was replaced with the UCSC Gene track, which indicates the gene, transcript, and protein name for additional isoforms of this gene. The only MbMt- and muscle-hypomethylated sites (orange rectangle), the short, novel antisense transcript (C1orf145) partly overlapping OBSCN (asterisk), and the associated doublet of MbMt-hypermethylated sites (asterisk in middle and bottom of figure) are indicated. The bottom of the figure is an enlargement of the C1orf145 and OBSCN exon 2 – 4 region (chr1:228,399,165-228,402,226) illustrating that ESC, which had higher levels of antisense RNA in this region (yellow box), did not have methylation at these two MbMt-hypermethylated sites in OBSCN exon 4 unlike Mb and Mt. The lower part of the figure also demonstrates differential methylation in Mb and Mt vs. skeletal muscle. Only Mt displayed H3K36me3 signal throughout the OBSCN gene region (not shown). 6 Figure S5. DNA methylation and chromatin epigenetic profiles of MYH7B/MIR499 suggest that differential splicing for this gene is controlled by myogenesis-associated DNA hypomethylation. (a) MYH7B displays significant skeletal muscle-associated hypomethylation in three intragenic regions, all of which also had low DNA methylation in cardiac muscle. The 5’ end of the gene had significant hypomethylation in Mb and Mt (not shown) and was also less methylated in skeletal muscle than in most other tissues (belowbut did not reach the p <0.01 level of significance. The entire MYH7B gene, the overlapping MIR499 gene, and the adjacent 5’ end of GSS and 3’ end of TRPC4AP genes are shown (chr20:33,542,729-33,591,014). Subregions with clusters of significant skeletal muscle-hypomethylated sites are boxed in the tracks with RRBS data. 7 Figure S5 (b). The only myogenic hypomethylation in MYH7B near moderate enrichment of transcription- promoting H3 modifications was the cluster of sites at exons 28 – 29. This cluster (orange boxes in differential methylation and H3K4Me1 or H3K4me2 tracks) also is near one of two myogenesis-associated CTCF binding sites (ENCODE/Broad, orange boxes in the CTCF binding track). Epigenetic marks for the same region as in Panel (a). There was no H3K9Me3 signal over most of this gene in both myogenic and nonmyogenic cells (not shown). A CTCF binding site that was prominent only in Mb and Mt is boxed in green. 8 References 1. McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, Wenger AM, Bejerano G. GREAT improves functional interpretation of cis-regulatory regions. Nature biotechnology 2010; 28:495-501. 2. Paylor R, Glaser B, Mupo A, Ataliotis P, Spencer C, Sobotka A, Sparks C, Choi CH, Oghalai J, Curran S, Murphy KC, Monks S, Williams N, O'Donovan MC, Owen MJ, Scambler PJ, Lindsay E. Tbx1 haploinsufficiency is linked to behavioral disorders in mice and humans: implications for 22q11 deletion syndrome. Proc Natl Acad Sci U S A 2006; 103:7729-34. 3. Emerich DF, Schneider P, Bintz B, Hudak J, Thanos CG. Aging reduces the neuroprotective capacity, VEGF secretion, and metabolic activity of rat choroid plexus epithelial cells. Cell Transplant 2007; 16:697-705. 9 Table S1. Cell cultures and tissues used for genome-wide profiling of methylation at CpG sites Number of Gender & age technical Unique sample number for Institutional Human cell culture or tissue type (abbreviation) Alternative ENCODE name Lineage of donor replicates ENCODEa sourceb Commercial or investigator source of initial cell culture Passaged cell cultures for the comparison of Mb plus Mt vs.