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

While studying myoblast methylomes and transcriptomes, we found that CDH15 had a remarkable preference for expression in both myoblasts and cerebellum. To understand how widespread such a relationship was and its epigenetic and biological correlates, we systematically looked for genes with similar transcription profiles and analyzed their DNA methylation and chromatin state and accessibility profiles in many different cell populations. Twenty genes were expressed preferentially in myoblasts and cerebellum (Myob/Cbl genes). Some shared DNA hypo- or hypermethylated regions in myoblasts and cerebellum. Particularly striking was ZNF556, whose promoter is hypomethylated in expressing cells but highly methylated in the many cell populations that do not express the gene. In reporter gene assays, we demonstrated that its promoter's activity is methylation sensitive. The atypical epigenetics of ZNF556 may have originated from its promoter's hypomethylation and selective activation in sperm progenitors and oocytes. Five of the Myob/Cbl genes (KCNJ12, ST8SIA5, ZIC1, VAX2, and EN2) have much higher RNA levels in cerebellum than in myoblasts and displayed myoblast-specific hypermethylation upstream and/or downstream of their promoters that may downmodulate expression. Differential DNA methylation was associated with alternative promoter usage for Myob/Cbl genes MCF2L, DOK7, CNPY1, and ANK1. Myob/Cbl genes PAX3, LBX1, ZNF556, ZIC1, EN2, and VAX2 encode sequence-specific transcription factors, which likely help drive the myoblast and cerebellum specificity of other Myob/Cbl genes. This study extends our understanding of epigenetic/transcription associations related to differentiation and may help elucidate relationships between epigenetic signatures and muscular dystrophies or cerebellar-linked neuropathologies.

Multivariate analysis of food consumption profiles in crisis settings.

Preventing malnutrition is one of the primary objectives of many humanitarian agencies, and household surveys are regularly employed to monitor food insecurity caused by political, economic, or environmental crises. Consumption frequencies for standard food groups are often collected to characterize the depth of food insecurity in a community and measure the impact of food assistance programs, producing a vector of bounded, correlated counts for each household. While aggregate indicators are typically used to summarize these results with a single statistic, they can be difficult to interpret and provide insufficient detail to judge the effectiveness of assistance programs. To address these limitations, we have developed a multivariate modeling framework for consumption frequency data. We introduce methods to update baseline models for the analysis of the smaller and more variable surveys typically collected in crisis settings, and we present an application of our approach to national consumption data collected in Yemen in 2014 and 2016 by the World Food Programme. The approach provides more nuanced and interpretable information about consumption changes in response to shocks and the effectiveness of humanitarian assistance.

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

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.

Evaluating differences in Young's Modulus of regenerated and uninjured mouse digit bone through microCT density-based calculation and nanoindentation testing.

The mouse digit tip amputation model is an excellent model of bone regeneration, but its size and shape present an obstacle for biomechanical testing. As a result, assessing the structural quality of the regenerated bone in this model has focused on mineral density and bone architecture analysis. Here we describe an image-processing based method for assessment of mechanical properties in the regenerated digit by using micro-computed tomography mineral density data to calculate spatially discrete Young's modulus values throughout the entire distal third phalange. Further, we validate this method through comparison to nanoindentation-measured values for Young's modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower Young's modulus compared to the uninjured digit, with a similar trend for experimental hardness values. Importantly, this method heightens the utility of the digit regeneration model, allows for more impactful treatment evaluation using the model, and introduces an analysis platform that can be used for other bones that do not conform to a standard long-bone model.

Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration.

De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Age-Dependent Changes in Bone Architecture, Patterning, and Biomechanics During Skeletal Regeneration.

Mouse digit amputation provides a useful model of bone growth after injury, in that the injury promotes intramembranous bone formation in an adult animal. The digit tip is composed of skin, nerves, blood vessels, bones, and tendons, all of which regenerate after digit tip amputation, making it a powerful model for multi-tissue regeneration. Bone integrity relies upon a balanced remodeling between bone resorption and formation, which, when disrupted, results in changes to bone architecture and biomechanics, particularly during aging. In this study, we used recently developed techniques to evaluate bone patterning differences between young and aged regenerated bone. This analysis suggests that aged mice have altered trabecular spacing and patterning and increased mineral density of the regenerated bone. To further characterize the biomechanics of regenerated bone, we measured elasticity using a micro-computed tomography image-processing method combined with nanoindentation. This analysis suggests that the regenerated bone demonstrates decreased elasticity compared with the uninjured bone, but there is no significant difference in elasticity between aged and young regenerated bone. These data highlight distinct architectural and biomechanical differences in regenerated bone in both young and aged mice and provide a new analysis tool for the digit amputation model to aid in evaluating the outcomes for potential therapeutic treatments to promote regeneration.

Epigenetics and expression of key genes associated with cardiac fibrosis: NLRP3, MMP2, MMP9, CCN2/CTGF and AGT.

Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix drive cardiac fibrosis and require changes in gene expression. Materials and methods: Using bioinformatics, both tissue-specific expression profiles and epigenomic profiles of some genes critical for cardiac fibrosis were examined, namely, NLRP3, MMP2, MMP9, CCN2/CTGF, AGT (encodes angiotensin II precursors) and hsa-mir-223 (post-transcriptionally regulates NLRP3). Results: In monocytes, neutrophils, fibroblasts, venous cells, liver and brain, enhancers or super-enhancers were found that correlate with high expression of these genes. One enhancer extended into a silent gene neighbor. These enhancers harbored tissue-specific foci of DNA hypomethylation, open chromatin and transcription factor binding. Conclusions: This study identified previously undescribed enhancers containing hypomethylated transcription factor binding subregions that are predicted to regulate expression of these cardiac fibrosis-inducing genes.

Plasma flow distal to tourniquet placement provides a physiological mechanism for tissue salvage.

Military literature has demonstrated the utility and safety of tourniquets in preventing mortality for some time, paving the way for increased use of tourniquets in civilian settings, including perioperatively to provide a bloodless surgical field. However, tourniquet use is not without risk and the subsequent effects of tissue ischemia can impede downstream rehabilitative efforts to regenerate and salvage nerve, muscle, tissue and bone in the limb. Limb ischemia studies in both the mouse and pig models have indicated not only that there is residual flow past the tourniquet by means of microcirculation, but also that recovery from tissue ischemia is dependent upon this microcirculation. Here we expand upon these previous studies using portable Near-Infrared Imaging to quantify residual plasma flow distal to the tourniquet in mice, pigs, and humans and leverage this flow to show that plasma can be supersaturated with oxygen to reduce intracellular hypoxia and promote tissue salvage following tourniquet placement. Our findings provide a mechanism of delivery for the application of oxygen, tissue preservation solutions, and anti-microbial agents prior to tourniquet release to improve postoperative recovery. In the current environment of increased tourniquet use, techniques which promote distal tissue preservation and limb salvage rates are crucial.

Epigenetics of Skeletal Muscle-Associated Genes in the ASB, LRRC, TMEM, and OSBPL Gene Families.

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, Leucine-Rich Repeat Containing (LRRC), Oxysterol Binding Protein Like (OSBPL), Ankyrin Repeat and Socs Box (ASB), and Transmembrane Protein (TMEM), 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). Some of these genes have associations with SkM or heart disease, cancer, bone disease, or other diseases. 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.

Does Fatigue Impact Static and Dynamic Balance Variables in Athletes with a Previous Ankle Injury?

Ankle injury, resulting in deficits in static and dynamic balance, can result in significant time loss to sport, affect daily activities and potentially place athletes at greater risk of re-injury. In order to identify athletes at risk of ankle injury accurate and reliable balance assessment tools are required. The purpose of the current study was to quantify reliability of static and dynamic balance variables in currently healthy, previously injured, athletes (n = 19) and assess the impact of an intense intermittent zig-zag running protocol to volitional exhaustion, rated by RPE, on balance variables. A test re-test design assessed short-term reliability and measurement error by computing ICC and 95% limits of agreement (LoA). The Y balance test was deemed a reliable measuring tool for assessing dynamic balance, recording strong reliability (ICC = 0.96, 95% LoA from -95.7 to 105.8%). A HURlabs iBalance force platform assessed the static balance variables sway velocity and C90area; sway velocity (mmˑs-1) recorded strong reliability (ICC = 0.79). Significant post-fatiguing protocol increases (p < 0.001) were detected in single-leg static balance for both C90area (mm2) and sway velocity (mmˑs-1) assessed on stable and unstable surfaces (stable: 227 ± 84 vs. 366 ± 146 mm2 and 18.6 ± 4.2 vs. 22.9 ± 5.3 mmˑs-1: unstable; 275 ± 128 vs. 370 ± 140 mm2 and 19.3 ± 4.3 vs. 21.5 ± 4.0 mmˑs-1). Non-significant post-fatiguing protocol differences (p > 0.05) were detected in dynamic balance variables (anterior, posteromedial, posterolateral and composite reach scores) measured at 4-min after completing the protocol. Further research should investigate the effects of fatigue on dynamic YBT variables immediately post-exercise and determine if differences exist when comparing previously injured and un-injured limbs.

Sirtuin 3 deficiency does not impede digit regeneration in mice.

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility-promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro. Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported. We show here, using the mouse digit amputation model, that SIRT3 deficiency has no impact on the regenerative capacity and architecture of bone and soft tissue. Regeneration occurs in SIRT3 deficient mice in spite of the reduced oxidative metabolic profile of the periosteal cells. These data suggest that bone regeneration, in contrast to homeostatic bone turnover, is not reliant upon active SIRT3, and our results highlight the need to examine known roles of SIRT3 in the context of injury.

Data showing atherosclerosis-associated differentially methylated regions are often at enhancers.

Atherosclerosis involves phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs). Data are given in tabular or figure format that illustrate genome-wide DNA methylation alterations in atherosclerotic vs. control aorta (athero DMRs). Data based upon publicly available chromatin state profiles are also shown for normal aorta, monocyte, and skeletal muscle tissue-specific DMRs and for aorta-specific chromatin features (enhancer chromatin, promoter chromatin, repressed chromatin, actively transcribed chromatin). Athero hypomethylated and hypermethylated DMRs as well as epigenetic and transcription profiles are described for the following genes: ACTA2, MYH10, MYH11 (SMC-associated genes); SMAD3 (a signaling gene for SMCs and other cell types); CD79B and SH3BP2 (leukocyte-associated genes); and TBX20 and genes in the HOXA, HOXB, HOXC, and HOXD clusters (T-box and homeobox developmental genes). The data reveal strong correlations between athero hypermethylated DMRs and regions of enhancer chromatin in aorta, which are discussed in the linked research article "Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers" (M. Lacey et al., 2019).

Expanding Research Capacity in Sub-Saharan Africa Through Informatics, Bioinformatics, and Data Science Training Programs in Mali.

Bioinformatics and data science research have boundless potential across Africa due to its high levels of genetic diversity and disproportionate burden of infectious diseases, including malaria, tuberculosis, HIV and AIDS, Ebola virus disease, and Lassa fever. This work lays out an incremental approach for reaching underserved countries in bioinformatics and data science research through a progression of capacity building, training, and research efforts. Two global health informatics training programs sponsored by the Fogarty International Center (FIC) were carried out at the University of Sciences, Techniques and Technologies of Bamako, Mali (USTTB) between 1999 and 2011. Together with capacity building efforts through the West Africa International Centers of Excellence in Malaria Research (ICEMR), this progress laid the groundwork for a bioinformatics and data science training program launched at USTTB as part of the Human Heredity and Health in Africa (H3Africa) initiative. Prior to the global health informatics training, its trainees published first or second authorship and third or higher authorship manuscripts at rates of 0.40 and 0.10 per year, respectively. Following the training, these rates increased to 0.70 and 1.23 per year, respectively, which was a statistically significant increase (p < 0.001). The bioinformatics and data science training program at USTTB commenced in 2017 focusing on student, faculty, and curriculum tiers of enhancement. The program's sustainable measures included institutional support for core elements, university tuition and fees, resource sharing and coordination with local research projects and companion training programs, increased student and faculty publication rates, and increased research proposal submissions. Challenges reliance of high-speed bandwidth availability on short-term funding, lack of a discounted software portal for basic software applications, protracted application processes for United States visas, lack of industry job positions, and low publication rates in the areas of bioinformatics and data science. Long-term, incremental processes are necessary for engaging historically underserved countries in bioinformatics and data science research. The multi-tiered enhancement approach laid out here provides a platform for generating bioinformatics and data science technicians, teachers, researchers, and program managers. Increased literature on bioinformatics and data science training approaches and progress is needed to provide a framework for establishing benchmarks on the topics.

Tissue-specific epigenetics of atherosclerosis-related ANGPT and ANGPTL genes.

AIM: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) genes (especially the five genes embedded in introns of host genes) and their association with atherosclerosis. METHODS: Transcription and epigenomic databases from various normal tissues were examined in the vicinity of ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. RESULTS: We identified tissue-specific enhancer chromatin regions that are likely to regulate transcription of ANGPT/ANGPTL genes and were intragenic, intergenic or host gene-linked. In addition, we found atherosclerosis-linked differentially methylated regions associated with ANGPT2 and with sequences encoding miR-145, a microRNA that targets ANGPT2 mRNA in cancers. CONCLUSION: Our findings implicate enhancers as major contributors to tissue-specific expression of ANGPT/ANGPTL genes, which play critical roles in angiogenesis, atherosclerosis, cancer, and inflammatory and metabolic diseases.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.

BACKGROUND AND AIMS: Atherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. METHODS: We examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. RESULTS: We found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis- and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. CONCLUSIONS: We provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription.

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Modeling Methylation Patterns with Long Read Sequencing Data.

Variation in cytosine methylation at CpG dinucleotides is often observed in genomic regions, and analysis typically focuses on estimating the proportion of methylated sites observed in a given region and comparing these levels across samples to determine association with conditions of interest. While sites are tacitly treated as independent, when observed at the level of individual molecules methylation patterns exhibit strong evidence of local spatial dependence. We previously developed a neighboring sites model to account for correlation and clustering behavior observed in two tandem repeat regions in a collection of ovarian carcinomas. We now introduce extensions of the model that account for the effect of distance between sites as well as asymmetric correlation in de novo methylation and demethylation rates. We apply our models to published data from a whole genome bisulfite sequencing experiment using long reads, estimating model parameters for a selection of CpG-dense regions spanning between 21 and 67 sites. Our methods detect evidence of local spatial correlation as a function of site-to-site distance and demonstrate the added value of employing long read sequencing data in epigenetic research.

Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression.

Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study-owing to their myogenic DMRs-overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage.