Biochemical and Biophysical Research Communications
Histone demethylase LSD1 is required to induce skeletal muscle differentiation by regulating myogenic factors
Research highlights
► LSD1 physically interacts with myogenic factors, MyoD and Mef2D on the target promoters. ► LSD1 regulates histone H3K9 methylation on myogenic promoters such as myogenin and MCK during myogenesis. ► Dysfunction of LSD1 impairs C2C12 myogenic differentiation.
Introduction
Skeletal muscle differentiation is a well-orchestrated process that is not only important in organogenesis, but also in postnatal growth and adult skeletal muscle regeneration. The series of events in myogenesis is tightly regulated by two major classes of transcription factors, Mef2 and MyoD [1]. Four isoforms of mammalian Mef2, Mef2A–D, share highly conserved N-terminal domains, MADS/Mef2 domain, which are responsible for DNA binding ability and protein–protein interaction [2]. Upon initiation of myogenesis, MyoD and Mef2 synergistically induce skeletal muscle differentiation via closely spaced DNA binding sites [3].
Transcription activity of MyoD and Mef2 is epigenetically regulated by histone methyltransferases (HMTs) and histone deacetylases (HDACs), which maintain gene silencing condition. In turn, histone acetyltransferases (HATs) uphold the active status of histone codes [4], [5], [6], [7]. Surprisingly, how the promoter region is epigenetically relieved from methylation prior to further acetylation has not been elucidated yet.
Epigenetic regulation, including post-translational modifications of histones, is crucial for differential gene expression during various cellular processes [8], [9]. Especially, the reversible methylation status of histone N-terminal tails provides an important regulatory mechanism in either gene activation or repression [10], [11], [12], [13], [14].
Lysine specific demethylase 1 (KDM1; LSD1; AOF2) is a histone demethylase with a dual substrate specificity for activation and repression markers, mono-, di-methyl group on H3K4 and H3K9 [15], [16], [17], [18]. Despite that LSD1 has been prominent in various cellular processes including differentiation, its role in skeletal muscle differentiation has not been explored yet [19], [20], [21].
In this study, it was attempted to determine if LSD1 is the histone demethylase involved in a skeletal muscle differentiation. In myoblast cell differentiation, LSD1 activates myogenic transcription factors. The epigenetic regulation by LSD1 provides insights into how myogenic transcription factors are relieved from a repressive status and are readily activated by acetyltransferases.
Section snippets
Cell culture and transient expression
C2C12 mouse myoblast cells were obtained from ATCC and cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum and antibiotics (growth medium, GM). For differentiation of C2C12 cells into myotubes, GM was replaced by media containing 2–5% horse serum with antibiotics (differentiation medium, DM). HEK293 cells were described previously [7] and were transfected using the calcium phosphate coprecipitation methods or Welfect-EX™ PLUS reagent (WelGENE, Korea).
DNA constructs
HA-Mef2D and
LSD1 is differentially expressed during myogenesis
During skeletal myogenic process, in which epigenetic regulation is critical in regulatory mechanism, any demethylase function has not yet been elucidated, despite of an evident necessity. Meanwhile, a histone demethylase, LSD1, has been reported to function in several developmental processes [20], [21], [26]; however, an involvement of LSD1 in skeletal muscle differentiation has not been suggested. Thus, we attempted to determine if LSD1 epigenetically regulates skeletal muscle differentiation
Discussion
The tight regulation of temporal gene expression during myogenic differentiation is fulfilled by various histone modifiers, which alter the chromatin structures [30]. Transcription activities of myogenic factors, MyoD and Mef2, are repressed by histone methyltransferases in myoblast [5], [6], [7]. Upon stimulus to differentiate into myotubes, however, transcriptional machines are turned on by recruiting coactivator complex including HATs, which acetylate the lysine residues that were previously
Acknowledgments
This work was supported by the grants from the Korea Healthcare Technology R&D Project (A090281), and National R&D Program for Cancer Control (0720460), Ministry of Health, Welfare & Family Affairs, and National Research Foundation Grant (NRF-2010-0018896) to H.D.Y., and Korean Research Foundation Grant (KRF-C00257) to H.D.Y. and E.J.C. J.C. was supported by Seoul Science Fellowship.
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