Promoter-adjacent DNA hypermethylation can downmodulate gene expression:TBX15in 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 en-zymatic 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 hyper-methylation, 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 en-hancer/extended promoter activity, thereby downmodulating, but not silencing, expression in myoblasts and certain kinds of skeletal muscle. This promoter-border hypermethylation is not found in cell types with a silent TBX15 gene probably because they have no transcription to modulate. 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.

Comparative computational analysis to distinguish mesenchymal stem cells from fibroblasts

“…Lower methylation patterns in MSCs were also detected for the VGLL4 gene, a co-transcriptional regulator of stem cell survival while fostering the colony formation capacity via interaction with the Rho/Rock pathway ( 86 ). In contrast, higher methylation of the T-Box transcription factor 15 in MSCs together with the generally more repressive role of T-box domain transcription factors could resemble a fibroblast-specific phenomenon as lower methylation values of the TBX15 gene might account for increased transcriptionally active TBX15 preventing fibroblasts from a myoblast phenotype ( 87 , 88 ).…”