Promoter-Adjacent DNA Hypermethylation Can Downmodulate Gene Expression: TBX15 in the Muscle Lineage

Abstract

TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection. These cloned regions exhibited strong promoter activity and, when placed upstream of a weak promoter, strong enhancer activity specifically in myoblast host cells. In vitro CpG methylation targeted to the DMR sequences in the plasmids resulted in 86–100% loss of promoter or enhancer activity, depending on the insert sequence. These results as well as chromatin epigenetic and transcription profiles for this gene in various cell types support the hypothesis that DNA hypermethylation immediately upstream and downstream of the unmethylated promoter region suppresses enhancer/extended promoter activity, thereby downmodulating, but not silencing, expression in myoblasts and certain kinds of skeletal muscle. This promoter-border hypermethylation was not found in cell types with a silent TBX15 gene, and these cells, instead, exhibit repressive chromatin in and around the promoter. TBX18, TBX2, TBX3 and TBX1 display TBX15-like hypermethylated DMRs at their promoter borders and preferential expression in myoblasts. Therefore, promoter-adjacent DNA hypermethylation for downmodulating transcription to prevent overexpression may be used more frequently for transcription regulation than currently appreciated.

Epigenetics of Genes Preferentially Expressed in Dissimilar Cell Populations: Myoblasts and Cerebellum

“…Myob, myoblasts; Cbl, cerebellum; hypom, hypomethylation; hyperm, hypermethylation. b Number of DMRs; the promoter hypomethylation in myoblasts for CDH15 was seen as a long specific LMR (Figure 1C); the cryptic promoter activity of the intragenic CDH15 hyperm DMR was previously reported [17]. c Prom, promoter; txn, transcription; ↑, upregulates; ↓, downmodulates; alt, alternative; Enh, enhancer chromatin; dnstm, downstream; upstm, upstream; adj, adjacent; repr chrom, repressive chromatin.…”

“…The DMRs probably downmodulate gene expression in myoblasts vs. in cerebellum. In a previous study of TBX15, a TF-encoding gene preferentially expressed in myoblasts and SkM but not in any brain region, we used reporter gene assays and in vitro methylation to demonstrate that enhancer and promoter activity of Myob-hyperm DMR sequences upstream or downstream of its TSS was strongly suppressed by DNA methylation [17]. In the case of PAX3, myoblast and cerebellum hypermethylation upstream and downstream of its promoter in both myoblasts and cerebellum is probably helping to keep transcription low in both cell populations (Table 1, Figure 7).…”

“…It is part of a methylation-sensitive cryptic promoter highly active in myoblast host cells, but not in MCF7 (breast cancer cell line). With the recent availability of our myoblast WGBS/EM-seq-derived myoblast DMRs [17] and a cerebellum WGBS profile from Loyfer et al [18], we re-examined the epigenetics of CDH15. It is not only preferentially but also strongly expressed in both cerebellum and myoblasts, and to a lesser extent in skeletal muscle (SkM; Figure 1A and Table 1).…”

“…The chromatin epigenomics data (chromatin state segmentation and H3K27ac) were from the Roadmap Epigenomics Project [19], as previously described [17], and visualized in the UCSC Genome Browser (https://www.genome.ucsc.edu/, accessed on 28 August 2023). Coordinates are in hg19, unless otherwise stated; tracks only in hg38 coordinates were lifted over to hg19.…”

“…We first identified twenty human genes that are preferentially expressed in myoblasts and cerebellum (Myob/Cbl genes). We then investigated transcriptional and epigenetic relationships for Myob/Cbl genes using our newly available whole-genome bisulfite sequencing (WGBS) or enzymatic methyl-seq (EM-seq) myoblast methylomes [17], a recent WGBS profile for cerebellum neurons from Loyfer et al [18], and chromatin epigenomics databases [19]. Unlike RRBS, which covers only up to 5% of the CpGs [20], WGBS and EM-seq allow the quantitation of methylation at essentially all the CpGs in the genome.…”