Epigenetics of skeletal muscle-associated genes in theASB, LRRC, TMEM, andOSBPLgene families

Abstract

Much remains to be discovered about the intersection of tissue-specific transcription control and the epigenetics of skeletal muscle (SkM), a very complex and dynamic organ. From four gene families, ASB, LRRC, TMEM, and OSBPL, we chose 21 genes that are preferentially expressed in human SkM relative to 52 other tissue types and analyzed relationships between their tissue-specific epigenetics and expression. We also compared their genetics, proteomics, and descriptions in the literature. For this study, we identified genes with little or no previous descriptions of SkM functionality (ASB4, ASB8, ASB10, ASB12, ASB16, LRRC14B, LRRC20, LRRC30, TMEM52, TMEM233, OSBPL6/ORP6, and OSBPL11/ORP11) and included genes whose SkM functions had been previously addressed (ASB2, ASB5, ASB11, ASB15, LRRC2, LRRC38, LRRC39, TMEM38A/TRIC-A, and TMEM38B/TRIC-B). Among the transcription-related SkM epigenetic features that we identified were super-enhancers, promoter DNA hypomethylation, lengthening of constitutive low-methylated promoter regions, and SkM-related enhancers for one gene embedded in a neighboring gene (e.g., ASB8-PFKM, LRRC39-DBT, and LRRC14B-PLEKHG4B gene-pairs). In addition, highly or lowly co-expressed long non-coding RNA (lncRNA) genes probably regulate several of these genes. Our findings give insights into tissue-specific epigenetic patterns and functionality of related genes in a gene family and can elucidate normal and disease-related regulation of gene expression in SkM.

Epigenetics of Muscle- and Brain-Specific Expression of KLHL Family Genes

“…These exceptions to generalizations about higher expression being associated with higher intragenic DNA methylation [ 4 ] and enhancer DNA hypomethylation usually being limited to foci [ 15 ] illustrate the importance of examining epigenetic profiles of individual genes to gain insight into their transcription regulation. As has been found for some other gene families [ 16 ], in the KLHL gene family, tissue-specific enhancer chromatin was more common than tissue-specific promoter chromatin. About half of these genes displayed SkM- or brain-associated demethylation extending upstream and/or downstream from a constitutively unmethylated CGI at the promoter region.…”

“…The most dramatic enhancer cooperation is seen in genes with super-enhancers. Super-enhancers are large structures consisting of clusters of neighboring enhancers (or enhancers and promoters) that usually are associated with strongly upregulating expression of differentiation-related genes [ 27 ], including SkM-associated genes [ 15 , 16 ]. Seven of the 17 SkM- or brain-associated KLHL family genes exhibited expression-linked super-enhancers ( Table 3 ).…”

“…The importance of having normal levels of expression of KLHL family genes is supported by small interfering RNA (siRNA) knockdown or overexpression studies in which decreases or increases in protein levels of the KLHL family genes impacted normal cellular function (e.g., for KLHL40 , KLHL41 , KEAP1 , ENC1, and KBTBD11 [ 30 , 63 , 64 , 65 , 66 ]). Chromatin and DNA epigenetics are frequently involved in setting or maintaining gene expression levels [ 4 , 16 , 62 ]. In addition, fine-tuning of RNA abundance by downregulation at the post-transcriptional level is often mediated by miRNAs.…”

“…The quantitation of cell culture RNA-seq data employed the Cufflinks tool as previously described [ 69 , 70 ]. For comparisons of RNA levels in myoblasts and myotubes, we used our previously generated data [ 16 ].…”

“…Enhancer chromatin in genome-wide studies is usually defined as regions enriched in histone H3 lysine 27 acetylation (H3K27ac) and histone H3K4 monomethylation (H3K4me1) while active promoters exhibit H3K27ac enrichment and H3K4 trimethylation (H3K4me3) instead of H3K4me1 and are usually adjacent to the TSS. Enhancers can be immediately upstream of promoters but are frequently found elsewhere [ 4 , 16 ]. Just as tissue-specific or disease-associated DNA methylation can down-regulate promoters, it can also down-modulate enhancers, mostly by limiting the accessibility and/or binding of transcription factors to the enhancer [ 15 , 17 ].…”