Systematic Analysis of Known and Candidate Lysine Demethylases in the Regulation of Myoblast Differentiation

https://doi.org/10.1016/j.jmb.2016.10.004Get rights and content

Highlights

  • LSD1 and MLL4 have opposing functions at Runx2 enhancer.

  • LSD1 suppresses Runx2 expression to promote myogenic differentiation.

  • Runx2 deletion rescues LSD1 deletion in myogenic differentiation.

Abstract

Histone methylation dynamics plays a critical role in cellular programming during development. For example, specific lysine methyltransferases (KMTs) and lysine demethylases (KDMs) have been implicated in the differentiation of mesenchymal stem cells into various cell lineages. However, a systematic and functional analysis for an entire family of KMT or KDM enzymes has not been performed. Here, we test the function of all the known and candidate KDMs in myoblast and osteoblast differentiation using the C2C12 cell differentiation model system. Our analysis identified that LSD1 is the only KDM required for myogenic differentiation and that KDM3B, KDM6A, and KDM8 are the candidate KDMs required for osteoblast differentiation. We find that LSD1, via H3K4me1 demethylation, represses the master regulator of osteoblast differentiation RUNX2 to promote myogenesis in the C2C12 model system. Finally, MLL4 is required for efficient osteoblast differentiation in part by countering LSD1 H3K4me1 demethylation at the RUNX2 enhancer. Together, our findings provide additional mechanisms by which lysine methylation signaling impacts on cell fate decisions.

Introduction

Chromatin regulation through the modification of histones plays a critical role during the differentiation of stem and progenitor cells. Changes in histone modifications at critical gene regulatory regions such as promoters and enhancers help mediate cell-type-specific gene expression programs [1]. In this context, active promoters are marked by histone H3 lysine 4 trimethylation (H3K4me3) whereas simultaneous H3 lysine 27 trimethylation (H3K27me3) is linked to lower expression of developmental genes in embryonic stem cells [2]. Enhancers are marked by H3 lysine 4 monomethylation (H3K4me1) [3] and H3K27 acetylation (H3K27ac) [4]. H3K27ac at enhancer is correlated with proximal gene expression, and its absence is linked to inactive enhancers.

Adult tissue stem cells and progenitor cells are important to maintain or repair adult tissue. Mesenchymal stem cells are present in adult tissue such as bone marrow and adipose tissue and can differentiate to osteocytes, adipocytes, and chondrocytes in vitro. Extensive evidence has demonstrated that differentiation of adult tissue stem cells and progenitor cells is regulated by histone methylation [5]. For example, the H3K27 methyltransferase EZH2 inhibits osteogenic differentiation, whereas the H3K27 demethylases KDM 6A and KDM6B enhance the osteogenic differentiation of mesenchymal stem cells [6], [7].

Muscle progenitor cells are bipotent and can differentiate not only to myotubes but also to osteogenic cells [8], [9]. In adult tissue, these bipotent progenitor cells may contribute to the repair of damaged muscle and bone fracture [10]. Both KMTs and KDMs have been implicated in myotube differentiation, and consistently, genome-wide studies have revealed dynamic changes in histone modification states during myotube differentiation [11], [12]. However, the full extent by which histone modifiers regulate differentiation of myoblasts is not clear. Mammalian skeletal myogenesis happens during early embryonic development and in response to damaged mature muscle. During myogenesis, myoblast, which arises from embryonic progenitor cells, differentiate into myocytes, which differentiate to form myotubes. This process is regulated by key transcription factors, including Pax3, Pax7, MEF, Myf5, MyoD, Mrf4, and Runx2, and these factors are in turn regulated by different chromatin factors [5]. For example, the KMTs G9a and EZH2 modulate both MyoD and MEF transcription during myogenesis [5], and the KDM LSD1 is required for myogenesis [1]. However, the extent by which the KDMs affect myogenesis and the extent by which the specific underlying molecular mechanism of enzymes like LSD1 influences this process are not completely understood.

In this study, we aimed to identify KDMs that regulate the differentiation of muscle progenitor cells. To this end, we knocked out all known and candidate KDMs in the mouse genome with cluster regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and examined myogenic and osteogenic differentiation using the C2C12 cell differentiation model system. Our analysis identified the H3K4 demethylase LSD1 as the only KDM required for myogenic differentiation via regulation of the expression of myogenic differentiation factors. These activities are mediated via LSD1 demethylation of H3K4me1 at the enhancer region of RUNX2, an osteogenic master regulator that inhibits myotube differentiation. We also identify MLL4, an H3K4 methyltransferase, as a positive regulator of myoblast osteogenic differentiation. Together, these results provide additional insights into the role of histone methylation in the regulation of stem cell differentiation.

Section snippets

Screen of known and candidate KDMs in the regulation of cellular potency in a model system

Mouse myoblast C2C12 cells are multipotent and can be differentiated into various cellular lineages including myotubules and osteoblasts [8]. We used this well-established cellular system to explore a role for the known and candidate protein KDMs and the related JmjC domain-containing hydroxylases in myogenic and osteogenic differentiation. The CRISPR/Cas9 system was utilized to delete genes in C2C12 cells. As Cas9 could not be efficiently transduced in C2C12 cells when co-expressed with small

Discussion

In this study, we identified LSD1 as a regulator of myogenic differentiation in the C2C12 model cell system. Although previous study reported that LSD1 induced myotube differentiation by demethylating H3K9me2 at the promoter of myogenic genes [14], only the neuron-specific isoform can demethylate H3K9me2 in vitro [23]. Here, we have provided evidence that the relevant substrates of LSD1 in myoblast-like cells are H3K4me1 and H3K4me2. Based on public ChIP-Seq data [11], [24], we found a large

Cell, plasmids, and antibodies

C2C12 (cat. no. CRL-1772) was purchased from American Type Culture Collection. pCW-Cas9 and pLX-sgRNA were gifts from Eric Lander and David Sabatini (Addgene plasmid #50661 and #50662). Anti-LSD1 antibody (Cell Signaling Technology; cat. no. 2139), anti-beta-tubulin antibody (Millipore; cat. no. 05-661), anti-lamin B antibody (Santa Cruz; cat. no, SC-6217), and anti-RUNX2 antibody (MBL, cat. no. D130-3) were used for Western blot. Anti-LSD1 antibody (Cell Signaling Technology; cat. no. 2184),

Acknowledgments

This work was supported in part by a grant from the NIH to O.G. (R01CA172560).

Conflict of Interest: O.G. is a co-founder of EpiCypher, Inc.

References (31)

  • K. Mousavi et al.

    eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci

    Mol. Cell

    (2013)
  • J.M. Dowen et al.

    Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes

    Cell

    (2014)
  • S. Chen et al.

    The PZP domain of AF10 senses unmodified H3K27 to regulate DOT1L-mediated methylation of H3K79

    Mol. Cell

    (2015)
  • T. Chen et al.

    Chromatin modifiers and remodellers: regulators of cellular differentiation

    Nat. Rev. Genet.

    (2014)
  • N.D. Heintzman et al.

    Histone modifications at human enhancers reflect global cell-type-specific gene expression

    Nature

    (2009)
  • Cited by (0)

    View full text