Roles of super enhancers and enhancer RNAs in skeletal muscle development and disease

ABSTRACT

Skeletal muscle development is a multistep biological process regulated by a variety of myogenic regulatory factors, including MyoG, MyoD, Myf5, and Myf6 (also known as MRF4), as well as members of the FoxO subfamily. Differentiation and regeneration during skeletal muscle myogenesis contribute to the physiological function of muscles. Super enhancers (SEs) and enhancer RNAs (eRNAs) are involved in the regulation of development and diseases. Few studies have identified the roles of SEs and eRNAs in muscle development and pathophysiology. To develop approaches to enhance skeletal muscle mass and function, a more comprehensive understanding of the key processes underlying muscular diseases is needed. In this review, we summarize the roles of SEs and eRNAs in muscle development and disease through affecting of DNA methylation, FoxO subfamily, RAS-MEK signaling, chromatin modifications and accessibility, MyoD and cis regulating target genes. The summary could inform strategies to increase muscle mass and treat muscle-related diseases.

KEYWORDS:

• Development
• differentiation
• enhancer RNAs
• skeletal muscle
• super enhancers

1. Introduction

Skeletal muscle is important for metabolism [1,2] and movement [3–5] and is the major contributor to body mass. In some pathological conditions, such as cancer cachexia [6,7], heart failure [8,9], Duchenne muscular dystrophy [10,11], burn injury [12,13], and rhabdomyosarcoma (RMS) [14,15], patients show muscle disorders. Skeletal muscle regeneration is a physiological process relying on the adult muscle stem cells (MuSCs) which are generally in a quiescent state. Following a muscle injury or disorders, MuSCs are activated, enter the cell cycle to proliferate, migrate to the impaired areas, enter the myogenic differentiation program differentiate, and eventually give rise to fusion forming newly myofibers [16]. The regulation of forming new myofibers from MuSCs during skeletal muscle development relies on a series of specific transcription factors. Pax3 and its paralog, Pax7, are considered to be markers of MuSCs, which support an essential role in the normal function of satellite cells in adult skeletal muscle. Pax3 is necessary for maintaining the migration of the cells in the somite and their migration to the myogenic sites [17]. Knockdown or deletion of Pax7 results in growth arrest [18,19] and significantly impaired regeneration as indicated by substantial deposition of fibrotic and decreased formation of myofibers [20]. Myogenic regulatory factors (MRFs), including MyoG, MyoD, Myf5, and Myf6 (also known as MRF4) are well-known regulators of muscle development. MyoG plays a vital role in myogenesis and homeostasis [21]. MyoD and Myf5 primarily participate in muscle specification and trigger the transformation of non-muscle cells into muscle cells, such as fibroblasts and chondroblasts [22,23]. MyoG and Myf6 function later in myogenesis, promoting the formation and maturation of myotubes [24,25]. Mice with mutations of the MyoG gene survive fetal development, but die immediately after birth due to failure to develop normal muscle mass [26]. Four members of the FoxO subfamily, FoxO1, FoxO3a, FoxO4, and FoxO6, are involved in diseases, such as diabetes [27], alveolar RMS [28], senescence [29], and cancer [30,31].

Next-generation sequencing technologies have facilitated comprehensive analysis of coding sequences and led to the discovery of numerous regulatory elements that do not act as templates for coding proteins [32]. In recent years, several studies have indicated that non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs), play vital roles in skeletal muscle development and disease, by acting as competitive endogenous RNA [33,34], regulating transcription factors, and participating in the epigenetic control of DNA methylation or chromatin activity [35–37]. Two systematic reviews by our groups summarized the known functions and mechanisms of lncRNAs and circRNAs in skeletal muscle development, as well the roles of miRNAs and lncRNAs in regulating the skeletal muscle atrophy seen in cachexia [38,39].

Among non-coding regulatory elements, super enhancers (SEs) were discovered at a relatively late stage. Enhancers, which were first shown to enhance the transcription of β-globin in rabbits in 1981 [40], are short regulatory elements in DNA that help activate the transcription of target genes remotely from gene locus. Whyte found that the master transcription factors of embryonic stem cells could bind special enhancers essential for maintaining the pluripotent state in 2013; these special enhancers were defined as SEs [41]. Compared with typical enhancers, SEs, which occur in large numbers of enhancer clusters, exhibit remarkably increased occupancy of transcription factors and RNAP II, enhance hypersensitivity to DNase I, and promote higher levels of H3K4me1 and H3K27ac density, as well as cohesin, Brd4, mediators, and p300/CBP occupancy [41–43]. High binding levels of the master transcription factors OCT4 and SOX2 are seen in enhancers near genes that determine embryonic stem cell identity [41]. Super and typical enhancers can be identified and distinguished from each other by sequencing nascent RNA, similar to traditional ChIP-seq [44]. In 2010, enhancer regions were found to actively transcribed [45,46]. The product of this transcription was termed enhancer RNA (eRNA). The production of eRNA requires three steps: recruitment of necessary transcription factors and coactivators; histone modifications at enhancer loci; and transcription, elongation, and processing of eRNA [47]. An increasing number of studies have suggested various roles for SEs and eRNAs in regulating multiple aspects of cell function in cancer [48,49], metabolic [50,51], immune diseases [52,53], Huntington disease [54] and Alzheimer [55], as well as skeletal muscle development and RMS which is a soft tissue tumor linked with the skeletal muscle lineage generally occurring in childhood and adolescence [56,57].

The discovery of SEs and eRNAs paved the way for modern cellular genomics, although their roles in regulating skeletal muscle development and pathophysiology remain unclear. In this review, we summarize the SEs and eRNAs that regulate skeletal muscle development according to recent reports. A better understanding of the functional mechanisms of SEs and eRNAs will aid efforts to increase muscle mass and could reveal potential therapeutic targets for muscle-related diseases.

2. Roles of SEs in skeletal muscle development and disease

Although the roles of coding sequences in muscle development have been extensively investigated, the key processes underlying the non-coding sequences driving muscle development and disease require further clarification. Recent reports have shown that SEs play roles in skeletal muscle development (Figure 1) and disease (Figure 2) via different mechanisms.

Figure 1. Schematic showing the role of SEs in skeletal muscle development. the various SEs that regulate myoblast differentiation are in green font.

Figure 2. Schematic showing the role of SEs in skeletal muscle disease. the various SEs that regulate disease are in green font.

SE activation can be affected by DNA hypomethylation, demethylation, and methylation. The main epigenetic modifications are histone and DNA modifications, which play an important role in the development of skeletal muscle. Preferential gene expression in skeletal muscle was associated with overlapping foci of DNA hypomethylation, according to bioinformatic analysis of 44 muscle-associated genes; half of these genes have skeletal muscle-specific SEs [58], consistent with a study revealing that tissue-specific regulatory genes of skeletal muscle are highly enriched in SEs [59]. A 0.258-kb distal enhancer of MyoD is completely unmethylated at all the CG sites tested in myogenic cells and a subpopulation of somite cells [60]. Recently, transfection assays confirmed the tissue specificity of the 0.3-kb core enhancer (CE) within the SE of MyoD in C2C12, and demonstrated its repression by methylation of three CG dinucleotides. DNA hypomethylation has been shown to increase enhancer tissue-specificity [58], while methylation of three CG nucleotide pairs in the MyoD CE strongly repressed its activity [58]. Myogenic stem cells display enhanced DNA methylation, while muscle differentiation was associated with a loss of DNA methylation in CG-poor regions. DNA methylation could prevent the deleterious activation of Myf5 [61]. Activation of the Myf5 SE was driven by DNA demethylation exclusively in muscle-committed cells, resulting in gene expression. Furthermore, upstream stimulatory factor 1 (Usf1) binding to the Myf5 SE occurs upon DNA demethylation in myogenic stem cells [61]. These results emphasize the function of DNA demethylation in lineage-specific activation of the muscle-identity gene Myf5 SE, in combination with the transcription factor Usf1, during cellular development [61]. Studies of epigenetic modifications via SEs during skeletal muscle development are needed.

SEs exert effects in myogenesis or RMS via members of the FoxO subfamily. In a recent study, SEs were uncovered to gradually remodel during myogenic differentiation by ChIP-seq analysis of histone marks; SE hotspots are co-bound by master transcription factors and MyoD. In-depth dissection showed that MyoD/FoxO3 interaction drove hotspot formation and SE activation [62]. In addition to FoxO3, FoxO1 also affects SE activity. In a cancer of muscle lineage, fusion-positive RMS, a translocated FoxO1 SE, drives the oncogene PAX3-FoxO1 in the late stage of myogenesis. Disruption of FoxO1 SE reduced PAX3-FoxO1 at the transcript and protein levels. PAX3 activates MyoD by binding and activating MyoD SEs [63], and several FoxO1 cis-regulatory domains interact with PAX3. HiChIP of H3K27ac revealed connections among the PAX3 promoter, additional intra-domain enhancers, and FoxO1 SE [63]. PAX3-FoxO1 can also reprogram the cis-regulatory landscape by eliciting SEs; PAX3-FoxO1 established auto-regulatory circuitry with transcription factors MyoG and MyoD via SEs. In cell lines and primary tumors, this myogenic SE loop is consistently seen [64]. These studies suggest a vital role of SEs in myogenesis and RMS.

SEs also play a role in muscle-related diseases by modulating RAS-MEK signaling. RMS is a common muscle-related disease arising from myoblasts, muscle stem cells, and activated satellite cells, as indicated by both in vivo and in vitro studies [58,61,62,65]. Ras-Raf-MEK-ERK-MAPK (RAS-MEK) is a signaling cascade that blocks myogenic differentiation [61,65]. In a recent report, a SE was found on the CASZ1 gene locus that occurs in human skeletal muscle myotubes but not myoblasts, which suggests that the increased expression of CASZ1 was driven by a SE during human skeletal myogenesis. Loss of CASZ1 in normal muscle cells led to a decrease in SE signaling at MyoD and MyoG [56]. CASZ1 induces differentiation of skeletal muscle and PAX-FoxO1 fusion-negative embryonal RMS (ERMS) by forming a feedforward loop with MyoG and MyoD. Overexpression of CASZ1 decreased Myf5 mRNA levels, and increased MyoD and MyoG mRNA levels. Analysis of the ChIP-seq data suggested that, in C2C12 myotubes, both MyoD and MyoG bind the promoter and enhancer regions of CASZ1. Silencing of MyoD or MyoG, but not Myf5, in C2C12 cells decreased CASZ1 expression, indicating MyoD and MyoG directly regulate CASZ1 transcription [56]. CASZ1 is inhibited by activated RAS-MEK signaling in ERMS with a RAS mutation [56]. These data indicate that restoration of CASZ1 in ERMS establishes new SEs. Another report showed that MEK inhibition can induce MyoG via the RAS-MEK effector pathway. MyoG subsequently opens chromatin and establishes SEs in genes required for late myogenic differentiation in RAS-driven RMS [65], suggesting that SEs function in RMS via RAS-MEK signaling.

3. eRNAs in skeletal muscle development

Enhancer-mediated regulation of various biological pathways and diseases has been extensively investigated [66–68]. eRNAs, as products transcribed from enhancers or SEs, have only recently been investigated; nevertheless, they have attracted attention in terms of their functions in skeletal muscle development and disease (Figure 3).

Figure 3. Schematic showing the role of eRNAs in skeletal muscle development. the various eRNAs that regulate the differentiation and regeneration of myoblasts are in green font.

Emerging evidence supports regulatory roles of eRNAs in chromatin modifications and accessibility during muscle cell differentiation [69,70]. Two enhancers, namely the CE and the distal regulatory region (DRR), can regulate the expression of MyoD [71–73]. The CE enhancer directs gene expression in somitic precursors during embryogenesis and the DRR enhancer does so in early differentiated myogenic cells [74]. The ^DRReRNAs and ^CEeRNAs are transcribed from the enhancer regions of the master regulator MyoD. ^CEeRNA facilitates chromatin restructuring in MyoD, resulting in polymerase II (Pol II) recruitment and expression. Upon MyoD gene activation and during differentiation, ^DRReRNA promotes the chromatin modifications at MyoG necessary for its expression, resulting in a feedforward loop for the myogenic program [70]. Thus, eRNAs direct gene activation by modulating chromatin remodeling at distinct regulatory genomic regions. ^DRReRNA is also recruited to the MyoG locus, where it colocalizes with MyoG nascent transcripts [69]. ^DRReRNA associates with the cohesin complex; depletion of cohesin or ^DRReRNA can decrease chromatin accessibility, thereby blocking MyoG activation and preventing muscle cell differentiation [69]. Thus, ^DRReRNA ensures spatially appropriate cohesin loading in trans, and regulates the expression of genes linked to muscle cell differentiation by decreasing chromatin accessibility. ^CEeRNA promotes chromatin restructuring in MyoD, resulting in Pol II recruitment and the expression, and regulation of transcription of the adjacent MyoD gene in cis [70,75].

MyoD plays a significant role in skeletal muscle development. eRNAs function in muscle development via regulation of MyoD. Lysine-specific demethylase 1 (LSD1) is a monoamine oxidase that can demethylate mono- or dimethylated lysine 4 and 9 of the histone H3 N-terminus (H3K4me1, H3K4me2 and H3K9me1, H3K9me2), thus promoting transcriptional repression or activation [76–79]. LSD1 is recruited to the MyoD CE, where it facilitates transcription of an eRNA that controls the timing of MyoD expression during myoblast commitment [75]. In myoblasts, depletion of lysine-specific LSD1 prevents recruitment of RNA Pol II and removal of H3K9 methylation on the CE, thereby precluding transcription of the non-coding eRNAs (^CEeRNAs) required for MyoD expression; this has been shown to occur in muscle progenitor cells during embryogenesis [75]. The effects of LSD1 inactivation can be efficiently reversed by the expression of the ^CEeRNA (−strand). MUNC, which is situated in the DRR enhancer of MyoD and formally classified as an eRNA (^DRR eRNA), is required for optimal myogenic differentiation, and induces specific myogenic transcripts, such as MyoD and MyoG, in trans [80]. Although MUNC is an eRNA of MyoD, it is also a trans-acting lncRNA whose sequence, structure, and co-operating factors include, but are not limited, to MyoD; as a lncRNA, MUNC regulates many myogenic genes [80,81]. Another seRNA PAM (Pax7 Associated Muscle lncRNA) which generated from a myoblast-specific SE also acts in trans, but without affecting MyoD. Timp2 is known to delay myogenic differentiation in C2C12 myoblasts [82]. Vim is an intermediate filament protein, which is also considered as a reliable marker for regenerating muscle tissue [83]. Studies indicate that seRNA PAM interacts with Ddx5 to tether PAM SE to its inter-chromosomal targets Timp2 and Vim to activate the gene expression, so as to promote satellite cell and C2C12 myoblast proliferation and regeneration [84].

In addition to MyoD, eRNAs regulate other target genes in cis. The SE-generated eRNAs seRNA-1 and seRNA-2 facilitate myogenic differentiation in vitro and in vivo. The master transcription factor MyoD is crucial for activating eRNA production. Knockdown of MyoD decreased myotube seRNAs generated from SEs, supporting a role of MyoD in seRNA induction during differentiation [57]. seRNA may bind and tether heterogeneous nuclear ribonucleoprotein L (hnRNPL) to the promoter of target myoglobin and apolipoprotein L6, to enhance transcription in cis by increasing the local concentration of Pol II or H3K36me3 [57]. When evaluating expression dynamics in vivo during injury-induced muscle regeneration, myoglobin and apolipoprotein L6 were highly expressed in homeostatic muscles, although this was markedly reduced after muscle injury, and continually elevated during regeneration [57], thereby supporting their role in promoting muscle regeneration. Disruption of the seRNA-1-hnRNPL interaction attenuates Pol II and H3K36me3 deposition at the myoglobin locus, thus reducing its transcription. Furthermore, analyses of hnRNPL binding transcriptome-wide have revealed that its association with eRNAs is a general phenomenon [57]. The eRNA–hnRNPL interaction may contribute to target mRNA activation.

The epigenomic reader Brd4 is also involved in regulating the production of eRNAs during cell differentiation and development. Brd4 is a member of the BET family; evidence from tissue-specific Brd4 knockout mice and derived cells indicates that Brd4 controls cell identity gene induction and is essential for myogenesis [85]. Brd4 colocalizes with lineage-determining transcription factors on active enhancers during differentiation. Deletion of Brd4 decreases eRNA production from Brd4+ adipogenic enhancers, cell identity gene induction, and cell differentiation [85]. The small-molecule competitive BET inhibitor JQ1 inhibits Brd4 binding, leading to displacement of the mediator complex and Pol II from enhancers, which in turn suppresses eRNA production and associated gene expression [86–88]. These discoveries suggest Brd4 is an enhancer epigenomic reader that mediates the relationship between eRNA and cell identity gene induction during differentiation.

4. Concluding remarks

Muscle development is a complex biological process strictly regulated by various pathways that also play a major role in related diseases. Dysregulation of these pathways is mediated by genetic and epigenetic alterations of protein coding genes and non-coding regulatory elements, which have been shown to function in myogenesis and related diseases.

Targeting disease-specific SEs may be a useful approach for identifying candidate biomarkers and therapeutic targets. Brd4, a member of the BET family, is essential for myogenesis. Brd4 inhibitors are promising candidate drugs for treating SE-related diseases, such as multiple myeloma and lymphoma [89,90]; the pharmacological efficacy and safety of these inhibitors are currently being evaluated in phase I clinical trials. Multiple myeloma cells were the first cells used to demonstrate how BET inhibitors can abolish SEs [86]. Young and Bradner, as pioneering researchers of SEs [86,91,92], collaborated with Syros Pharmaceuticals (SYRS) to research and develop SE-related targets for cancer treatment. We expect that further SE research will provide an in-depth understanding of molecular pathogenesis and progression, and aid the development of novel therapies to treat skeletal muscle disease.

A broad range of disease-associated mutations occur outside the coding genome; multiple genome-wide association studies have indicated that many of these mutations occur in enhancers or their transcribed RNAs [93,94]. eRNAs are useful for “mapping” anxiety disorders [95], cardiac hypertrophy [96], lymphoblast proliferation [97], neurological disorders [98], recurrent pregnancy loss [99,100], and cancer [101–104]. The inhibition of hypoxia-inducible enhancer RNA (HERNA) 1 blocked stress-induced cardiac pathogenesis and significantly improved the overall survival of diseased mice, thereby demonstrating the therapeutic potential of eRNAs [96]. Although therapeutic applications are still in their infancy, drugs affecting eRNAs directly or indirectly will likely be useful for treating cancer and muscle-related diseases in the future.

Skeletal muscle development is a complex progress regulated by various MRFs, which constitute the core of the transcriptional cascades that induce myogenesis and muscle phenotypes. Next-generation sequencing has led to the discovery of numerous non-coding regulatory elements, which play significant roles in skeletal muscle development and disease via different mechanisms. Research on preventing or treating muscle-related diseases, among other diseases, through targeting SEs and eRNAs is still in its infancy. Nevertheless, drugs targeting SEs and eRNAs may emerge as important options for treating muscle-related diseases.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing not applicable – no new data generated.

Additional information

Funding

This work was supported by grants from the Guangdong Basic and Applied Basic Research Foundation (grant number 2021A1515010862), the Science and Technology Program of Guangzhou (grant numbers 202102080042, 202102080224), the Administration of Traditional Chinese Medicine of Guangdong Province (grant numbers 20211020), the Medical Scientific Research Foundation of Guangdong Province (grant number A2022216, B2021221), and the Science Foundation of Guangdong Second Provincial General Hospital (grant number 3D-B2020015).