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Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes*

  • Alexandra A. Lolis
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
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  • Priya Londhe
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
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  • Benjamin C. Beggs
    Affiliations
    Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
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  • Stephanie D. Byrum
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Science, Little Rock, Arkansas 72205
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  • Alan J. Tackett
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Science, Little Rock, Arkansas 72205
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  • Judith K. Davie
    Correspondence
    To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University School of Medicine, 229 Neckers Building, 1245 Lincoln Dr., Carbondale, IL 62901. Tel.: 618-453-5002; Fax: 618-453-6440;
    Affiliations
    Department of Biochemistry and Molecular Biology and Simmons Cancer Institute, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institute of Health Grants R01DA025755, F32GM093614, UL1RR029884, P30GM103450, and P20GM103429.
    1 Both authors contributed equally to this work.
Open AccessPublished:January 30, 2013DOI:https://doi.org/10.1074/jbc.M112.426718
      Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell.

      Introduction

      Gene regulation in skeletal muscle is controlled by a family of highly related basic helix-loop-helix transcription factors, the myogenic regulatory factors (MRFs).
      The abbreviations used are: MRF
      myogenic regulatory factor
      RNAPII
      RNA polymerase II
      TAP
      tandem affinity purification.
      The MRF family includes Myf5, MyoD, myogenin, and Myf6 (also known as MRF4). The MRFs dimerize with E proteins and bind E box sequences (CANNTG) in the regulatory regions of muscle genes (
      • Berkes C.A.
      • Tapscott S.J.
      MyoD and the transcriptional control of myogenesis.
      ). The MRFs work in conjunction with the MEF2 family of MADS box transcription factors (
      • Blais A.
      • Tsikitis M.
      • Acosta-Alvear D.
      • Sharan R.
      • Kluger Y.
      • Dynlacht B.D.
      An initial blueprint for myogenic differentiation.
      ). MEF2 proteins alone do not have myogenic activity but synergize with the MRFs to enhance gene expression during myogenesis (
      • Molkentin J.D.
      • Black B.L.
      • Martin J.F.
      • Olson E.N.
      Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins.
      ,
      • Wang D.Z.
      • Valdez M.R.
      • McAnally J.
      • Richardson J.
      • Olson E.N.
      The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development.
      ).
      The MRFs play overlapping but non-redundant roles in myogenesis. As revealed by mouse knockouts, Myf5 and MyoD function early in myogenesis to confer a myogenic fate on mesodermal progenitor cells (
      • Rudnicki M.A.
      • Schnegelsberg P.N.
      • Stead R.H.
      • Braun T.
      • Arnold H.H.
      • Jaenisch R.
      MyoD or Myf-5 is required for the formation of skeletal muscle.
      ). Myf6 has roles in both early and late events in myogenesis (
      • Kassar-Duchossoy L.
      • Gayraud-Morel B.
      • Gomès D.
      • Rocancourt D.
      • Buckingham M.
      • Shinin V.
      • Tajbakhsh S.
      Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.
      ). Myogenin functions later in myogenesis to stimulate specified myoblasts to differentiate into functional myofibers. Null mutations in myogenin cause lethality, making it unique among the MRFs (
      • Hasty P.
      • Bradley A.
      • Morris J.H.
      • Edmondson D.G.
      • Venuti J.M.
      • Olson E.N.
      • Klein W.H.
      Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.
      ,
      • Nabeshima Y.
      • Hanaoka K.
      • Hayasaka M.
      • Esumi E.
      • Li S.
      • Nonaka I.
      • Nabeshima Y.
      Myogenin gene disruption results in perinatal lethality because of severe muscle defect.
      ). In myogenin-null mice, myoblasts are specified, but muscle fibers form poorly (
      • Venuti J.M.
      • Morris J.H.
      • Vivian J.L.
      • Olson E.N.
      • Klein W.H.
      Myogenin is required for late but not early aspects of myogenesis during mouse development.
      ).
      Chromatin modifications and chromatin-modifying enzymes have been shown to play essential roles with the MRFs in establishing required transcriptional control during myogenesis. MyoD can remodel chromatin and activate transcription from silent loci much more efficiently than myogenin (
      • Gerber A.N.
      • Klesert T.R.
      • Bergstrom D.A.
      • Tapscott S.J.
      Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin. A mechanism for lineage determination in myogenesis.
      ). Acetyltransferases interact with MyoD and acetylate promoter histones as well as MyoD itself (reviewed in Ref.
      • Berkes C.A.
      • Tapscott S.J.
      MyoD and the transcriptional control of myogenesis.
      ). The activity of the MEF2 proteins is controlled by interactions with the class II histone deacetylases, which function to repress differentiation (reviewed in Ref.
      • Potthoff M.J.
      • Olson E.N.
      MEF2. A central regulator of diverse developmental programs.
      ). The ATP-dependent chromatin-remodeling enzyme SWI/SNF interacts with MyoD, myogenin, and MEF2 proteins and is required for muscle differentiation via its ability to alter chromatin structure at endogenous, muscle-specific loci (
      • de la Serna I.L.
      • Carlson K.A.
      • Imbalzano A.N.
      Mammalian SWI/SNF complexes promote MyoD-mediated muscle differentiation.
      ,
      • de la Serna I.L.
      • Ohkawa Y.
      • Berkes C.A.
      • Bergstrom D.A.
      • Dacwag C.S.
      • Tapscott S.J.
      • Imbalzano A.N.
      MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.
      ,
      • Simone C.
      • Forcales S.V.
      • Hill D.A.
      • Imbalzano A.N.
      • Latella L.
      • Puri P.L.
      p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci.
      ,
      • Ohkawa Y.
      • Marfella C.G.
      • Imbalzano A.N.
      Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1.
      ,
      • Ohkawa Y.
      • Yoshimura S.
      • Higashi C.
      • Marfella C.G.
      • Dacwag C.S.
      • Tachibana T.
      • Imbalzano A.N.
      Myogenin and the SWI/SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis.
      ).
      The facilitates chromatin transcription (FACT) complex was first identified as a complex required for transcript elongation on nucleosomal templates. It is a histone chaperone that is crucial for nucleosomal reorganization during transcription (
      • Orphanides G.
      • LeRoy G.
      • Chang C.H.
      • Luse D.S.
      • Reinberg D.
      FACT, a factor that facilitates transcript elongation through nucleosomes.
      ,
      • Orphanides G.
      • Wu W.H.
      • Lane W.S.
      • Hampsey M.
      • Reinberg D.
      The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.
      ), DNA replication (
      • Wittmeyer J.
      • Joss L.
      • Formosa T.
      Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha.
      ,
      • Wittmeyer J.
      • Formosa T.
      The Saccharomyces cerevisiae DNA polymerase α catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein.
      ), and DNA repair (
      • Bruhn S.L.
      • Pil P.M.
      • Essigmann J.M.
      • Housman D.E.
      • Lippard S.J.
      Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.
      ,
      • Yarnell A.T.
      • Oh S.
      • Reinberg D.
      • Lippard S.J.
      Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin.
      ). FACT acts by inducing either a structural change or stabilizing an existing alternative nucleosomal structure (
      • Winkler D.D.
      • Muthurajan U.M.
      • Hieb A.R.
      • Luger K.
      Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events.
      ,
      • Winkler D.D.
      • Luger K.
      The histone chaperone FACT. Structural insights and mechanisms for nucleosome reorganization.
      ), an activity termed “nucleosomal reorganization” (
      • Formosa T.
      FACT and the reorganized nucleosome.
      ). FACT has a high affinity for H2A-H2B dimers and can induce displacement of these dimers from nucleosomes in vitro (
      • Belotserkovskaya R.
      • Oh S.
      • Bondarenko V.A.
      • Orphanides G.
      • Studitsky V.M.
      • Reinberg D.
      FACT facilitates transcription-dependent nucleosome alteration.
      ,
      • Xin H.
      • Takahata S.
      • Blanksma M.
      • McCullough L.
      • Stillman D.J.
      • Formosa T.
      yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement.
      ).
      Mammalian FACT is made up of two subunits, SSRP1 (structure-specific recognition protein 1) and SPT16 (suppressor of Ty 16), which have both shown to be essential for nucleosomal reorganization (
      • Orphanides G.
      • Wu W.H.
      • Lane W.S.
      • Hampsey M.
      • Reinberg D.
      The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.
      ,
      • Winkler D.D.
      • Luger K.
      The histone chaperone FACT. Structural insights and mechanisms for nucleosome reorganization.
      ). FACT promotes transcription by RNAPII by several mechanisms. In the yeast Saccharomyces cerevisiae, FACT has been shown to be required for the eviction of histones from the GAL1–10 and PHO5 promoters (
      • Xin H.
      • Takahata S.
      • Blanksma M.
      • McCullough L.
      • Stillman D.J.
      • Formosa T.
      yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement.
      ,
      • Ransom M.
      • Williams S.K.
      • Dechassa M.L.
      • Das C.
      • Linger J.
      • Adkins M.
      • Liu C.
      • Bartholomew B.
      • Tyler J.K.
      FACT and the proteasome promote promoter chromatin disassembly and transcriptional initiation.
      ), and it promotes binding of the TATA-binding protein (TBP) to chromatin (
      • Biswas D.
      • Yu Y.
      • Prall M.
      • Formosa T.
      • Stillman D.J.
      The yeast FACT complex has a role in transcriptional initiation.
      ). FACT has also been shown to travel with elongating RNAPII and promote efficient elongation (
      • Mason P.B.
      • Struhl K.
      The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo.
      ). Less is understood about the function of FACT in mammalian cells, but several studies have implicated FACT in promoting muscle-specific gene expression. In smooth muscle, FACT has been seen to interact with MKL1, where it synergistically activates its transcriptional activity (
      • Kihara T.
      • Kano F.
      • Murata M.
      Modulation of SRF-dependent gene expression by association of SPT16 with MKL1.
      ). MKL1 is expressed in a variety of different cell types preceding cellular differentiation (
      • Spencer J.A.
      • Baron M.H.
      • Olson E.N.
      Cooperative transcriptional activation by serum response factor and the high mobility group protein SSRP1.
      ) and functions as an activator of serum response factor, a MADS box transcription factor that is responsible for controlling genes involved in cell proliferation and differentiation. It has also been shown that SSRP1 interacts with serum response factor, thus leading to an increase in DNA binding activity of serum response factor and an increased activation of its promoters, implying that SSRP1 is a coregulator of serum response factor-dependent transcription (
      • Spencer J.A.
      • Baron M.H.
      • Olson E.N.
      Cooperative transcriptional activation by serum response factor and the high mobility group protein SSRP1.
      ).
      We have discovered that the FACT complex interacts with myogenin and promotes differentiation-specific muscle gene expression. Although the physical interaction is restricted to myogenin, FACT is highly stimulatory for myogenin-dependent gene expression in the presence of both myogenin and MyoD. FACT is recruited to muscle-specific genes upon differentiation through the interaction with myogenin and travels with RNA polymerase II during elongation of these genes. Nucleosomes are depleted at these promoters coincident with the arrival of FACT, and we show that SSRP1 is required for this depletion and for expression of differentiation-specific genes. This work aids in our understanding of the factors that control the differentiation program of skeletal muscle and advances our knowledge of the role and recruitment of FACT in skeletal muscle.

      DISCUSSION

      We have found that myogenin interacts with the FACT complex. The interaction with myogenin recruits FACT to muscle-specific genes bound by myogenin, where FACT promotes nucleosome disassembly at promoters, thus promoting gene expression. Our data suggest that FACT plays a role in removing nucleosomes from muscle-specific promoter regions to allow the general transcription machinery to access the DNA sequence. A role for FACT in promoting chromatin disassembly from promoters has been shown for the PHO5 promoter in yeast, but our work is the first to show this in mammalian cells for muscle-specific genes. The activity of histone disassembly at promoters is distinct from the well established role of FACT in chromatin reassembly behind an elongating RNAPII. A promoter-specific role for FACT in activating gene expression has been suggested by studies that showed that FACT is required for TBP recruitment to the GAL1 and HO promoters (
      • Mason P.B.
      • Struhl K.
      The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo.
      ,
      • Biswas D.
      • Dutta-Biswas R.
      • Mitra D.
      • Shibata Y.
      • Strahl B.D.
      • Formosa T.
      • Stillman D.J.
      Opposing roles for Set2 and yFACT in regulating TBP binding at promoters.
      ). These dual processes link the process of initiation and elongation (
      • Mason P.B.
      • Struhl K.
      The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo.
      ).
      We show here the novel finding that FACT is required for efficient promoter activation of mammalian muscle differentiation-specific genes. Given this role, it was initially surprising that FACT was down-regulated as these genes were activated. However, we show that the role of FACT appears to be in the initial stages of promoter disassembly and activation. Once nucleosomes are removed from the promoter and the genes are active, we hypothesize that FACT is no longer needed to maintain gene expression at these promoters. The observed down-regulation of SPT16 and, to a lesser extent, SSRP1, is consistent with prior data showing that FACT is expressed in proliferating cells and down-regulated in differentiated cells (
      • Garcia H.
      • Fleyshman D.
      • Kolesnikova K.
      • Safina A.
      • Commane M.
      • Paszkiewicz G.
      • Omelian A.
      • Morrison C.
      • Gurova K.
      Expression of FACT in mammalian tissues suggests its role in maintaining of undifferentiated state of cells.
      ). However, we have also observed that SPT16 is not down-regulated as rapidly in primary myoblasts as in C2C12 cells. This result again strengthens the importance of the role we describe here for FACT in establishing the differentiation program in skeletal muscle.
      The specific interaction of FACT with myogenin and not MyoD is intriguing, as it suggests that the recruitment of FACT may be one mechanism that myogenin uses to initiate gene expression. However, it is clear from our data that the presence of both MyoD and myogenin leads to the highest activation in the presence of FACT. MyoD is a more robust transactivator than myogenin and has been shown to be bound to many of the promoters occupied by myogenin (
      • Davie J.K.
      • Cho J.H.
      • Meadows E.
      • Flynn J.M.
      • Knapp J.R.
      • Klein W.H.
      Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.
      ,
      • Londhe P.
      • Davie J.K.
      Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes.
      ,
      • Cao Y.
      • Yao Z.
      • Sarkar D.
      • Lawrence M.
      • Sanchez G.J.
      • Parker M.H.
      • MacQuarrie K.L.
      • Davison J.
      • Morgan M.T.
      • Ruzzo W.L.
      • Gentleman R.C.
      • Tapscott S.J.
      Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming.
      ,
      • Tai P.W.
      • Fisher-Aylor K.I.
      • Himeda C.L.
      • Smith C.L.
      • Mackenzie A.P.
      • Helterline D.L.
      • Angello J.C.
      • Welikson R.E.
      • Wold B.J.
      • Hauschka S.D.
      Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer.
      ). Thus, it has been difficult to understand the unique role of myogenin at these genes. Clearly, many genes are dependent on the presence of myogenin in vivo, as the null mutation of myogenin results in lethality (
      • Hasty P.
      • Bradley A.
      • Morris J.H.
      • Edmondson D.G.
      • Venuti J.M.
      • Olson E.N.
      • Klein W.H.
      Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.
      ,
      • Nabeshima Y.
      • Hanaoka K.
      • Hayasaka M.
      • Esumi E.
      • Li S.
      • Nonaka I.
      • Nabeshima Y.
      Myogenin gene disruption results in perinatal lethality because of severe muscle defect.
      ).
      It has been shown that MyoD can activate genes in a repressed chromatin environment, whereas myogenin cannot (
      • Gerber A.N.
      • Klesert T.R.
      • Bergstrom D.A.
      • Tapscott S.J.
      Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin. A mechanism for lineage determination in myogenesis.
      ). This suggests that the interaction with FACT is not sufficient to enable myogenin to activate genes in a highly repressed chromatin environment. However, our data clearly show that the interaction with FACT is required for myogenin to activate target genes from an inactive state. This suggests that MyoD and myogenin cooperate to activate specific genes by independently recruiting distinct chromatin modification and remodeling complexes.
      Our data show that the interaction with myogenin recruits FACT to muscle-specific promoters, but we also show that FACT travels with the elongating RNAPII at these genes, consistent with the well established role of FACT in promoting elongation by reassembling nucleosomes in the wake of RNAPII. It has been shown recently that recruitment of the SSRP1 subunit of FACT coincides with expression in both myoblasts and myotubes (
      • Vethantham V.
      • Yang Y.
      • Bowman C.
      • Asp P.
      • Lee J.H.
      • Skalnik D.G.
      • Dynlacht B.D.
      Dynamic loss of H2B ubiquitylation without corresponding changes in H3K4 trimethylation during myogenic differentiation.
      ). In this study, robust association of SSRP1 was observed at Acta1, Trim63, Myog, and Myh3, genes up-regulated in myotubes that have all been shown to be regulated by myogenin. SSRP1 was also associated with genes expressed in myotubes that are not targets of myogenin, and the basis of this recruitment is not currently understood.
      In summary, we have shown here that FACT contributes to the initiation of transcription at muscle-specific genes. The works reveals a unique function of myogenin in recruiting the FACT complex to promote nucleosome disassembly and gene expression. Recent work has shown that BAF60c, a subunit of the SWI/SNF chromatin remodeling complex, facilitates MyoD binding to target genes (
      • Forcales S.V.
      • Albini S.
      • Giordani L.
      • Malecova B.
      • Cignolo L.
      • Chernov A.
      • Coutinho P.
      • Saccone V.
      • Consalvi S.
      • Williams R.
      • Wang K.
      • Wu Z.
      • Baranovskaya S.
      • Miller A.
      • Dilworth F.J.
      • Puri P.L.
      Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex.
      ). BAF60c phosphorylation, mediated by p38α/β, then recruits SWI/SNF to remodel chromatin and promote the binding of the MRFs to target genes (
      • de la Serna I.L.
      • Ohkawa Y.
      • Berkes C.A.
      • Bergstrom D.A.
      • Dacwag C.S.
      • Tapscott S.J.
      • Imbalzano A.N.
      MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.
      ). Our work suggests that the binding of myogenin then recruits the FACT complex, which acts to disrupt nucleosomes at the promoters to, presumably, promote recruitment of the general transcription machinery. The stepwise contribution of each of these factors remains to be determined, but it is clear that multiple chromatin modifying complexes are required to establish appropriate transcriptional control at muscle-specific genes. Future studies will be required to understand the precise order of events required to initiate gene expression at target genes and to establish the precise role of FACT in initiating transcription at myogenin-dependent genes.

      Acknowledgments

      We thank Takanori Kihara, Osaka University, and Masayuki Murata, University of Tokyo for the pCMV-4-SPT16 expression construct. We also thank Rhonda Bassel-Duby and Eric Olson (University of Texas Southwestern) for providing the TRE reporter and MEF2C expression plasmid, Shuang Zhang for cloning the N-TAP myogenin construct, Shuang Zhang and Meiling Zhang for construction and characterization of the stable N-TAP myogenin cell lines used for the TAP purification, Bo Zhu for assistance with the immunostaining protocol, Brian Heine for cloning of the myogenin truncation constructs, and the University of Arkansas for Medical Science Proteomics Facility for mass spectrometric support.

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