The Nuclear Receptor Corepressor N-CoR Regulates Differentiation: N-CoR Directly Interacts with MyoD
Peter Bailey, Michael Downes, Patrick Lau, Jonathan Harris, Shen Liang Chen, Yasuo Hamamori, Vittorio Sartorelli, and George E. O. Muscat University of Queensland (P.B., M.D., P.L., J.H., S.L.C., G.E.O.M.) Centre for Molecular and Cellular Biology Ritchie Research Laboratories, B402A St Lucia, 4072 Queensland, Australia University of Southern California (Y.H., V.S.) Institute of Genetic Medicine No. 140 Los Angeles, California 90033
Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAa/b), Mad/ Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell’s decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stim0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society
ulated the activity of MyoD by approximately 10fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis. (Molecular Endocrinology 13: 1155–1168, 1999)
INTRODUCTION The process of skeletal muscle differentiation involves the activation and induction of a large array of musclespecific genes. The myoD gene family encode myogenic specific basic helix loop helix (bHLH) proteins that activate and control this process (reviewed in Refs. 1–3). MyoD plays a dual role during myogenesis, activating both muscle-specific gene transcription and promoting cell cycle exit by inducing the expression of p21Cip-1/Waf-1, an inhibitor of cyclin-dependent kinases and cellular proliferation (4–6). Transactivation by MyoD involves 1) the bHLH domain, which is involved in both DNA binding and dimerization; 2) heterodimerization of MyoD with ubiquitously expressed E2A gene products, E12 and E47 (7); 3) the binding of MyoD-E2A heterodimers to specific E-box motifs (CANNTG) in muscle-specific enhancers (reviewed in Refs. 4–6); and 4) the recruitment of the cofactors, p300 and PCAF (p300/CBP associating factor) (8–10). p300 and PCAF are critical coactivators for MyoD during myogenic commitment and differentiation. The N-terminal acid-rich activation domain of MyoD directly interacts with p300 and recruits PCAF to form a ternary multimeric complex on promoter elements (8). It has been suggested that the formation of this com1155
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plex leads to hyperacetylated and transcriptionally permissive chromatin. Moreover, p300 and PCAF coactivate myoD-mediated transactivation of the p21 gene and are necessary for MyoD-mediated cell cycle arrest (11). Cofactors function as bridges between DNA-binding proteins and the basal transcriptional machinery. The identification of corepressors (i.e. N-CoR/RIP13 and SMRT/TRAC) that interact with the thyroid hormone receptor, retinoic acid receptor, and vitamin D receptor (TR, RAR, and VDR) has shed some light on the mechanism of transcriptional repression (by classical nuclear receptors) in the absence of ligand (12– 14). The C-terminal receptor interaction domain (RID) of the corepressors interacts with the ligand binding domain/DE region of unliganded receptors. This interaction induces a series of protein-protein interactions that repress transcription. The corepressors contain two interaction domains that interact independently and synergistically with the nuclear receptors (15, 16). These corepressors interact with Sin3 and recruit the histone deacetylases (HDAc-1 or Rpd-3/HDAc-2) that lead to hypoacetylation of the histones. This deacetylation leads to conformational changes that stabilize the nucleosome structure, and limit the accessibility of the chromatin to the transcriptional machinery (17–19). The N-CoR/Sin3/HDAc complex mediates transcriptional repression from a wide variety of other nonreceptor-mediated pathways including the bHLH-LZ proteins, Mad/Mxi, that mediate repression of myc activities and tumor suppression (18), retinoblastomamediated repression of E2F action (20, 21), and the oncoproteins PLZF-RARa (22, 23) and LAZ3/BCL6 (24), which are involved in acute promyelocytic leukemia and non-Hodgkin lymphomas, respectively. These receptor- and non-receptor-mediated pathways share common attributes: they are converted in response to environmental stimuli from the repressive to the operative condition and function in the management of differentiation and cell division. N-CoR and the splice variants (RIP13a and RIP13D1) mediate transcriptional repression by the orphan nuclear receptors, Rev-erbA (15, 25), RVR (15, 25), and chick ovalbumin upstream promoter transcription factor II (COUP-TF II) (26). The carboxy-terminal regions of N-CoR and RIP13 that encode the receptor interaction domains are almost identical. There is one major difference between the RIP13s and N-CoR. The first 1016 amino acids of N-CoR that encode repression domains 1 and 2 are replaced by 10 unique amino acids at the amino-terminal end of the RIP13s (Ref. 16 and Fig. 1). However, the RIP13s contains seven copies of a repeated motif, G-s-l-s/tq-G-t-P, which is associated with repressor activity. Interaction of the Rev-erb a/b orphan nuclear receptors with the corepressors requires an intact ligandbinding domain (15, 25, 26), and physical association between the corepressors and the nuclear receptors is dependent on two corepressor interaction regions, lo-
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cated in helix 3 and helix 11, that probably form a corepressor interface in three-dimensional space (25). The corepressor complexes that mediate repression of transcription by 1) the nuclear hormone receptors, 2) Mad/Mxi, 3) RB (retinoblastoma), and 4) the oncoproteins [PLZF and LAZ3/BCL6] have been characterized in terms of proteins that regulate chromatin architecture (17–21). Furthermore, notable progress in elucidating the central role of the corepressor complex (N-CoR/Sin3) in transcriptional repression has been observed (23, 24). These corepressors recruit the histone deacetylase complex (HDAc-1/-2 and several other polypeptides) that deacetylates nucleosomal histones, and lead to the reduced accessibility of the DNA to the transcriptional machinery via chromatin remodeling. Recent evidence suggests that N-CoR and Sin3 directly interact with the key components of the transcriptional initiation process (TFIIB, and the TBP-associating factors) to inhibit basal transcription in an alternative repression pathway (27, 28). The function of N-CoR and the multiprotein corepressor complex in the process of cellular differentiation, tissue-specific transcription, and coupled phenotypic acquisition remain to be resolved. This study focuses on the functional role of N-CoR during myogenesis, a well characterized paradigm of mammalian differentiation, and investigates the mechanisms used by N-CoR in the repression of MyoD activity (a master regulatory protein) and regulation of differentiation.
RESULTS N-CoR mRNA Is Repressed during Myogenic Differentiation The nuclear corepressor protein N-CoR has been shown to interact with a number of transcription factors and to actively repress basal level transcription. N-CoR mRNA is highly expressed in adult skeletal muscle. Two variant truncated forms of N-CoR, RIP13a and RIP13D1, have also been identified. RIP13a and RIP13D1 proteins have been implicated in orphan receptor (i.e. RVR and Rev-erbAa)-mediated repression as bona fide corepressor proteins, but their expression in skeletal muscle has not yet been investigated. To examine the expression levels of N-CoR, RIP13a, and RIP13D1 mRNA in muscle cells, Northern analysis of RNA isolated from proliferating C2C12 myoblast cells was undertaken. The cDNA probe used in the Northern analysis corresponded to the carboxy-terminal RID of N-CoR, a region that is highly conserved between all three transcripts (i.e. N-CoR, RIP13a, and RIP13D1; Fig. 1A). It was observed that only one ;9-kb mRNA transcript was expressed in proliferating C2C12 myoblasts. This ;9-kb band corresponds to N-CoR mRNA. The N-CoR variants, RIP13D1 and RIP13a, should produce mRNA transcripts around 4.5 kb (16); however, transcripts of this size were not
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Fig. 1. The N-CoR mRNA Is Repressed during Myogenesis A, Diagramatic representation of the nuclear corepressor protein, N-CoR, and its splice variants, RIP13WT and RIP13D1 (for retinoid X receptor interacting proteins). Striped areas in the N termini of RIP13 and RIP13D1 represent unique N-terminal regions. There are two distinct differences between RIP13a and N-CoR. The first 1016 amino acids of N-CoR that encode repression domains I and 2 are replaced by 10 unique amino acids at the amino-terminal end of RIP13a. Another difference is the replacement of 48 amino acids of N-CoR (aa 1235–1282) with a serine in RIP13a (aa 228). Several minor amino acid changes were described in detail by Seol et al. (4) The N-CoR and RIP13a RIDs are identical. Specifically, ID-I corresponded to the region between amino acids 2218 and 2451 of N-CoR [1], and to amino acids 1164–1397 in RIP13a [ID-I in N-CoR, RIP13a and RIP13D1 are identical]. Northern analysis indicates that C2 myogenic cells express N-CoR mRNA (;9Kb) but not RIP13WT or RIP13D1 mRNA (;4.5 Kb). RNA was isolated from proliferating C2C12 myoblast cells and probed with 32P-radiolabeled cDNA spanning corepressor receptor interaction domain composed of interaction domains I and II (ID I1II) of N-CoR. B, N-CoR mRNA levels are down-regulated during skeletal muscle differentiation. Total RNA was isolated from PMB (;50% confluent), CMB (100% confluent) cultured in growth medium, and from developing myotubes 4, 8, and 24 h after serum withdrawal (i.e. propogated in DM, differentiation medium). After blotting, RNA was probed with 32P-radiolabeled cDNA encoding 18S rRNA, MyoD, Myogenin, Id (inhibitor of differentiation), CylinD1, p21cip-1/waf-1, and N-CoR. GM, Growth medium (DMEM containing 20% FCS); DM, differentiation medium (DMEM containing 2% horse serum).The induction of myogenin and p21 mRNAs confirm that these cells have undergone terminal differentiation.
detected, indicating that C2C12 cells only express N-CoR mRNA (Fig. 1A) or that the RIP13s were very rarely expressed. To gain some insight into the role played by N-CoR during the process of myogenesis, we initially investigated the expression pattern of N-CoR mRNA during the conversion of mouse myoblast C2C12 cells into multinucleated myotubes. Proliferating C2C12 myoblasts were induced to biochemically and morphologically differentiate into postmitotic multinucleated myotubes by serum withdrawal in culture over a 24- to 72-h period. The transition from a nonmuscle phenotype to a contractile phenotype is associated with the activation/expression of 1) the myoD gene family (myoD, myogenin, myf-5, and MRF-4), and 2) a structurally diverse group of genes that encode a functional sarcomere responsible for contraction. Concomitant with these events is terminal cell cycle exit, characterized by the repression of cyclin D1 and the activation
of the cell cycle inhibitor, p21. Total RNA was isolated from proliferating myoblasts (PMB), confluent myoblasts (CMB), and postmitotic myotubes after 4, 8, and 24 h of serum withdrawal and examined by Northern blot analysis. N-CoR mRNA was abundantly expressed in myoblasts; however, this transcript is suppressed, relative to 18S rRNA, as myoblasts exit the cell cycle and fuse to form differentiated multinucleated myotubes that have acquired a muscle-specific phenotype (Fig. 1B). Down-regulation of N-CoR mRNA correlated with the recent observations that the mRNAs encoding the three orphan nuclear receptors, Rev-erbAa (29), RVR (30), and COUP-TF II (31), are repressed during myogenesis. These orphans have been demonstrated to antagonistically regulate myogenesis, repress myoD mRNA expression, abrogate the induction of myogenin and p21 mRNA after serum withdrawal, and interact with N-CoR to repress transcription (15, 25, 26). Concomitant with this decrease
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in N-CoR mRNA was the induction of myogenin and p21 mRNAs, relative to 18S rRNA, which confirmed that these cells had terminally differentiated (Fig. 1B). The repression of cyclin D1 and the inhibitor of differentiation (Id) (relative to the equivalent levels of 18S rRNA) confirmed that these cells were exiting the cell cycle. The expression of MyoD mRNA in myoblasts and myotubes confirmed the myogenic nature of these cells (Fig. 1B). The differential expression of N-CoR mRNA suggested that this cofactor may regulate the process of differentiation and/or the maintenance of the proliferative cycle. N-CoR Expression in Myogenic Cells Blocks the Induction of Myogenin and p21 mRNA and Suppresses MyoD mRNA The Northern analyses demonstrated that repression of the N-CoR mRNA correlate with the biochemical and morphological differentiation of myogenic cells, concomitant with the acquisition of a contractile phenotype. To further delineate the role played by N-CoR in myogenesis and to identify the target(s) of this corepressor in muscle, we proceeded to examine the effect of exogenous N-CoR expression on C2C12 cell differentiation. Hence, C2 muscle cells were cotransfected with plasmids encoding full-length N-CoR (pSG5-N-CoR) and the G418 resistance gene, Neomycin (pCMVNEO). Stable transfectants were then isolated as a polyclonal pool of G418-resistant colonies (comprised of .20 individual resistant colonies). For reference, this pool of cells is denoted as C2:N-CoR. To examine the effect of exogenous N-CoR expression on factors involved in determination (e.g. MyoD), cell cycle regulation (e.g. cyclin D1 and p21), and differentiation (myogenin), we again isolated total cytoplasmic RNA from C2:N-CoR and normal C2C12 cells before and 72 h after serum withdrawal. After blotting, the RNA was probed with 18S rRNA, MyoD, myogenin, cyclin D1, and p21-labeled cDNAs (Fig. 2A). Comparisons of C2:N-CoR to native C2C12 cells showed that the level of myoD mRNA in the presence and absence of serum was significantly reduced in the C2:N-CoR cell line. Furthermore, the induction of the p21 and myogenin mRNAs after serum withdrawal was completely ablated in these cells (Fig. 2A). Interestingly, exogenous expression of N-CoR did not effect the repression of cyclin D1 after serum withdrawal, relative to the equivalent levels of 18S rRNA (Fig. 2A). The lack of the tissue-specific bHLH master regulators, MyoD and myogenin, and the cyclin-dependent kinase inhibitor, p21Waf-1/Cip-1, in the C2:N-CoR cells after 72 h of serum withdrawal correlates with the inability of this cell line to differentiate morphologically (Fig. 2A). In summary, the data above suggest that N-CoR regulates the transcription/expression of the musclespecific HBH genes (that are auto-regulated by their own expression) and functions as a negative regulator of myogenic differentiation.
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N-CoR Inhibits myoD-Mediated Myogenic Conversion of Pluripotential C3H10T1/2 Cells MyoD directly interacts with other cofactors (e.g. the coactivators, p300 and PCAF) that regulate its activity (9, 11), and N-CoR functions as a corepressor for a similar class of transcription factors, the bHLH-zip proteins (e.g. Mad/Mxi-1) (18). These observations, in the context of our expression analysis, suggested to us that N-CoR may function to directly regulate the action of the muscle-specific bHLH protein, MyoD. We tested this hypothesis by investigating the impact of N-CoR expression on the MyoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. This system has been used to functionally demonstrate that concomitant expression of the coactivators, p300 and PCAF, with MyoD mediated more efficient myogenic conversion of pluripotent cells (relative to MyoD alone) (8, 9). In this system, transient transfection of a MyoD expression vector under the control of an SV40 promoter leads to the phenotypic/myogenic conversion of these cells as scored by immunostaining with a monoclonal antibody directed against the fast isoform of the major thick filament protein, skeletal myosin heavy chain (MHC). MHC is associated with the contractile phenotype and is expressed during the process of muscle differentiation. Please note that an identical SV40 promoter linked to the LUC reporter in the same vector was not affected/regulated by N-CoR expression. Hence, the observed effects of concomitant MyoD and N-CoR transfection are independent of repression of myoD expression. C3H10T1/2 cells were transiently transfected with pSG5-MyoD in combination with either pSG5 (vector/ vehicle only) or pSG5-N-CoR. As expected, cells transfected with pSG5-MyoD were found to be positively stained with the MHC antibody (Fig. 2B). In contrast, cells that were cotransfected with both MyoD and N-CoR did not undergo myogenic conversion (Fig. 2B). The experiments presented above suggest that the cofactor, N-CoR, is involved in the inhibition of myoD-mediated myogenic conversion of pluripotent C3H10T1/2 cells. This repression of MyoD action is independent of the silencing of its gene expression in this system; hence, we suggest that N-CoR directly regulates the action of MyoD by posttranscriptional mechanisms. In summary, we propose that N-CoR functions as corepressor of the myogenic specific, bHLH protein, MyoD. N-CoR Inhibits MyoD-Mediated Transactivation The experiments presented above suggest that NCoR directly regulates MyoD action in vivo by a mechanism that is independent of MyoD gene expression. To confirm this, we examined the effect of N-CoR expression on MyoD-mediated transactivation in the GAL4 hybrid system. In these assays the activity of MyoD is independent of its binding to its cognate binding motif, the E-Box (CANNTG). If N-CoR directly
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Fig. 2. Exogenous N-CoR Expression Ablates MyoD-Mediated Myogenesis A, Stable exogenous expression of N-CoR in C2 myogenic cells blocks biochemical differentiation by suppressing myoD mRNA levels, and blocking the induction of myogenin and p21cip-1/waf-1 mRNAs. Total RNA was isolated from both normal C2C12 and stably tansfected C2C12:N-CoR cells at the PMB stage (;50% confluent) cultured in growth medium, and at a stage 72 h after the withdrawal of serum (i.e. propagated in differentiation medium). RNA samples were blotted and probed with 32P-radiolabeled cDNA encoding 18S rRNA, MyoD, Myogenin, CylinD1, and p21cip-1/waf-1. GM, Growth medium (DMEM containing 20% FCS); DM, differentiation medium (DMEM containing 2% horse serum). B, N-CoR inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. C3H10T1/2 cells were transiently transfected with 3 mg of pSG5-MyoD in combination with either 3 mg of pSG5 alone or 3 mg of pSG5-N-CoR. After 4 days in 2% horse serum, cells were immunostained with a monoclonal antibody directed toward the fast isoform of the major thick filament protein, skeletal MHC. MHC-positive cells are stained red and nuclei counterstained blue by Mayer’s hematoxylin.
regulates MyoD transcriptional activity, then the potential of GAL4-MyoD to transactivated gene expression should be greatly reduced in this assay (Fig. 3A). C3H10T1/2 nonmuscle cells were cotransfected with GAL4-MyoD and the G5E1b-LUC reporter in the presence and absence of pSG5-N-CoR. G5E1b-LUC contains five copies of the GAL4-binding site placed upstream of a minimal E1b promoter. As expected, cotransfection of the GAL4-dependent reporter with Gal-MyoD alone very efficiently transactivated the expression of the LUC reporter gene (Fig. 3B). However, cotransfection of Gal-MyoD and increasing quantities of the pSG5 N-CoR expression vector significantly repressed (.5-fold) Gal-MyoD-mediated transactivation in a dose-dependent fashion (Fig. 3B). In contrast, pSG5-N-CoR had no effect on GAL0 expression. This result demonstrates that N-CoR is capable of silencing MyoD-mediated transactivation in a dose-dependent manner.
We similarly examined the ability of N-CoR to repress the activity of Gal4-MyoD in myogenic C2C12 cells. Again, N-CoR silenced GAL4-MyoDmediated transactivation (Fig. 3C). Subsequently, we investigated whether N-CoR could repress MyoD-dependent E-box-driven transcription in myogenic cells. To achieve this, we used a 4REtkLUC reporter construct, in which four copies of the right E-box of the muscle-specific MCK enhancer (a cognate binding site for MyoD) are placed upstream of a minimal thymidine kinase (tk) promoter linked to the LUC reporter gene. This reporter has been demonstrated to facilitate MyoD-dependent transactivation. C2C12 muscle cells were cotransfected with the 4RE-tkLUC reporter in combination with either the pSG5 or pSG5-N-CoR vectors. Cotransfection of C2C12 cells with pSG5-N-CoR significantly repressed (;5-fold) the expression of the 4RE promoter in myogenic cells Fig. 3D). In summary, we
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Fig. 3. N-CoR Inhibits MyoD-Mediated Transactivation A, Diagramatic representation of the GAL4 hybrid assay. This assay was used to determine the effect of N-CoR expression on MyoD transcriptional activity. Cells are transfected with a plasmid encoding GAL4-MyoD (i.e. the yeast GAL4 DNAbinding domain fused in frame with full-length MyoD) and a reporter construct in which five copies of the GAL4-binding site are placed upstream of the minimal E1b promoter (i.e. G5E1b-LUC). B, N-CoR inhibits MyoD-mediated transactivation in C3H10T1/2 nonmuscle cells. C, N-CoR inhibits MyoD-mediated transactivation in C2C12 muscle cells. D, N-CoR represses MyoD-dependent/E-box driven transcription in C2C12 muscle cells. Fold repression is expressed relative to LUC activity obtained after cotransfection with the 4RE-tkLUC reporter and pSG5 alone. Repression mediated by pSG5 alone is arbitarily set at 1. E, TSA treatment increases (;6 fold) GAL-MyoD-mediated transactivation of the G5E1b-LUC reporter in C2 myogenic cells. F, TSA treatment blocks the ability of N-CoR to repress the MyoD-dependent reporter, 4RE-tkLUC, in C2C12 cells.
observed that MyoD-mediated transactivation in the presence and absence of its cognate binding motif is silenced in muscle and nonmuscle cells by the cofactor, N-CoR. We hypothesized that N-CoR could function as a corepressor of MyoD activity/function. We used an alternative approach to confirming the functional role of the N-CoR complex in the regulation of MyoD activity and to gain insight into the mechanism of N-CoR-mediated repression. We examined the ability of the histone deacetylase inhibitor, trichostatin A (TSA, 100 ng/ml), to regulate MyoD-mediated transactivation. We initially examined the affect of TSA treatment on the activity of Gal-MyoD in myogenic C2 cells. We observed that TSA treatment increased the activity of MyoD by approximately 6-fold (Fig. 3E). Furthermore, consistent with the role of the corepressor-HDAc-1 complex in the regulation of MyoD activity, TSA treatment blocked the ability of N-CoR to repress the MyoD-dependent reporter, 4RE (Fig. 3F). Our data indicated that one mechanism by which NCoR represses MyoD-mediated and -dependent transcription involves the recruitment of histone deacetylases.
N-CoR Directly Interacts with MyoD: The AminoTerminal Repression Domain (RD1) of N-CoR Mediates the Interaction and Is Required for Repression of Myogenesis We postulated that N-CoR could be functioning to repress MyoD activity/function by directly interacting with MyoD. To determine this, we used the in vitro glutathione-S-transferase (GST) pull-down assay, in which glutathione agarose-immobilized GST and GST-MyoD proteins were incubated with in vitro 35Sradiolabeled full-length N-CoR (Fig. 4A). This assay clearly showed that N-CoR and MyoD directly interact in vitro (Fig. 4B). To map the region within N-CoR that is involved in mediating this interaction, we examined the ability of in vitro 35S-labeled repression domain [amino acids (aa) 1–1017] and receptor interaction domain (aa 1848– 2453) (Fig. 4B) to interact with the immobilized GSTMyoD fusion protein. It was found that amino acids 1–1017, which encode the repression domain, interact very efficiently with GST-MyoD (Fig. 4B). The receptor interaction domain also specifically interacts with GST-MyoD in vitro, although far less efficiently than
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Fig. 4. N-CoR Directly Interacts with MyoD The amino-terminal repression domain (RD1) of N-CoR mediates the interaction and is required for repression of myogenesis. A, Diagramatic representation showing the N-CoR-regulatory repression and receptor interaction domains. B, The repression domain of N-CoR interacts with MyoD in vitro. Glutathione agarose immobilized GST, and GST-MyoD proteins were incubated with either 35S-radiolabeled full-length N-CoR, N-CoR repression domain (RD, aa 1–1017), or N-CoR RID (aa 1848–2453). The input lanes represent approximately 10% of the total protein. C, Amino acids 1–465 that encompass repression domain 1 (RD1) of N-CoR are capable of mediating MyoD binding in vitro. N-CoR amino acids 1–465 and 1–703 were radiolabeled with [35S]methionine by in vitro transcription/translation and assayed for their ability to interact with glutathione-immobilized GST and GST-MyoD full length. The input lanes represent approximately 10% of the total protein. D, Exogenous expression of RIP13D1 (i.e. an N-CoR splice variant that lacks N-CoR repression domains RD1 and RD2) in C2 myogenic cells does not affect terminal differentiation. Total RNA was isolated form both normal C2C12 and stably tansfected C2C12:RIP13D1 cells at the PMB stage (;50% confluent), the CMB stage (100% confluent) grown in growth medium, and at stages 4, 8, 24, and 72 h after the withdrawal of serum grown in differentiation medium. RNA samples were blotted and probed with 32P-radiolabeled cDNA encoding 18S rRNA, MyoD, Myogenin, CylinD1, b-actin, p21cip-1/waf-1, and N-CoR. GM, Growth medium (DMEM containing 20% FCS); DM, differentiation medium (DMEM containing 2% horse serum).
the native protein and the repression domain. To delimit the region within the repressor domain that interacts with N-CoR, we examined the ability of two 35Slabeled fragments, encoding aa 1–703 and aa 1–465, to efficiently and directly interact with GST-MyoD (Fig. 4C). We observed that both fragments bound MyoD efficiently (relative to full-length native N-CoR). This clearly demonstrates that the region (1–465) encompassing repression domain 1 (RD1), encoded by aa 1–312, can facilitate the interaction with MyoD. To examine whether the repression domain of NCoR is critical for the regulation of myogenesis and bHLH gene expression in vivo, we examined the effect of exogenous RIP13D1 expression in muscle cells. RIP13D1 is an N-CoR variant that is almost identical to N-CoR, except it lacks the first 1017 amino acids of N-CoR, which encode the two amino-terminal repression domains and facilitate the interaction with MyoD. The biochemical profile of C2C12 cells that express exogenous RIP13D1 mRNA (denoted C2:RIP13D1) was compared with normal C2C12 cells via Northern analysis of mRNAs that encode the key regulators and
markers of myogenic differentiation. Total RNA from both C2:RIP13D1 and normal C2 cells was isolated from PMB, CMB, and myotubes after 4, 8, 24, and 72 h of serum withdrawal. RNA samples were blotted and probed with 32P-labeled cDNA encoding 18S rRNA, MyoD, Myogenin, Cylin D1, b-actin, p21cip-1/waf-1, and N-CoR. It should be noted that the C2:RIP13D1 cell line expresses the exogenous RIP1D1 mRNA at levels similar to or greater than the endogenous N-CoR transcript. When compared with normal C2C12 cells, the C2:RIP13D1 cells exhibited a similar biochemical profile of mRNA expression before and after serum withdrawal (Fig. 4D). Specifically, they expressed MyoD before and after serum withdrawal. The myogenin and p21 mRNAs were induced after serum withdrawal, whereas the N-CoR, b-actin, and cyclin D1 mRNAs were all repressed. This demonstrated that the phenotype of these cells was not affected by RIP13 expression. This strongly suggested that the affect of N-CoR on myogenic differentiation is mediated by the amino-terminal repression domain of the cofactor, which specifically interacts with MyoD. Furthermore,
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this region is lacking in the RIP13 variants, which have no affect on myogenesis after stable transfection, and suggests that the change in phenotype induced by N-CoR expression is not due to the selection process. In summary, the data suggest that the amino-terminal region of the corepressor, N-CoR, which encodes the repression domain, directly interacts with MyoD in a functional manner to regulate its activity during myogenesis. The bHLH Region of MyoD Mediates the Interaction with N-CoR: The bHLH Domain Functions as a Repressor To delimit the region within MyoD (see Fig. 5A) that interacts with N-CoR, we examined the ability of a number of GST-MyoD fusion chimaeras containing functional subdomains of MyoD and immobilized on glutathione agarose beads [i.e. GST-MyoD (aa 1–318), GST-N terminal MyoD (aa 1–100), GST-bHLH (102– 161), and GST-C terminal MyoD (aa 162–318)] to interact with the 35S-labeled N-CoR (see Fig. 5A). As expected, full-length MyoD interacted with N-CoR. The bHLH region of MyoD linked to GST also inter-
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acted strongly with in vitro translated N-CoR (Fig. 5A). In contrast, the GST-MyoD N-terminal and C-terminal regions did not support any significant interaction with N-CoR. This suggests that the bHLH domain of MyoD (aa 109–162) is required for N-CoR binding. GST pull-down analysis identified the bHLH domain of MyoD as the minimal functional domain capable of interacting with N-CoR in vitro. Hence, we examined whether the bHLH domain of MyoD acts as an independent repressor domain in the GAL4 hybrid system. We investigated the transcriptional activity of GalMyoD-bHLH relative to Gal-MyoD with respect to two GAL4-dependent reporters, G5E1b-LUC (Fig. 5, B and D) and MH100tk-LUC (Fig. 5, C and E) in two separate cell lines, C3H10T1/2 and JEG-3. The E1b and MH100 plasmids contain multiple GAL4-binding sites upstream of the basal adenoviral E1b and tk promoter linked to LUC, respectively. Both reporter constructs are in routine use in many laboratories (8, 17, 26); however, the tk-promoter has a higher background level of expression and is often used to identify repression domains. We initially cotransfected JEG-3 and C3H10T1/2 cells with the G5E1b-LUC construct
Fig. 5. The bHLH Region of MyoD Mediates the Interaction with N-CoR: The bHLH Domain Functions as a Repressor A, The bHLH region of MyoD interacts with N-CoR in vitro. Full-length N-CoR was radiolabeled with [35S]methionine by in vitro transcription/translation and assayed for its ability to interact with glutathione-immobilized GST, GST-MyoD full length, GSTMyoD N-terminal, GST-MyoD C-terminal, and GST-MyoD bHLH. The input lanes represent approximately 10% of the total protein. B, GAL-MyoD bHLH fails to transactivate the G5e1b-LUC reporter in C3H10T1/2 cells. C, GAL-MyoD bHLH represses (;2 fold) MH100-LUC reporter basal transcription in 10T1/2 cells. D, GAL-MyoD bHLH fails to transactivate the G5e1b-LUC reporter in JEG-3 cells. E, GAL-MyoD bHLH represses (;5 fold) the MH100-LUC reporter basal transcription in JEG-3 cells. F, Internal deletion of the basic or HLH region of MyoD compromises the ability to repress the MH100 reporter in JEG-3 cells. In panels B–F, C3H10T1/2 and JEG-3 cells were transfected with 1 mg of either G5E1b-LUC or MH100-LUC and 0.3 mg of either GAL0, GAL-MyoD, or GAL-bHLH. Cells were harvested 15 h after the addition of liposomes in 5% charcoal-stripped serum and assayed for luciferase activity. Fold repression is expressed relative to the LUC activity obtained after cotransfection of the MH100-LUC reporter with GAL0 alone. Repression mediated by GAL0 is arbitarily set at 1. G, TSA treatment blocks the ability of Gal-MyoD-bHLH to repress MH100LUC reporter basal transcription. Cells were transfected in the presence or absence of 100 ng/ml TSA.
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in combination with either Gal-MyoD or Gal-MyoDbHLH. The low level of basal transcription exhibited by this reporter facilitates the identification of transcriptionally active protein domains. As expected, GALMyoD efficiently activated transcription in both cell lines. In contrast, GAL-MyoD bHLH did not exhibit any transcriptional activity in either C3H10T1/2 or JEG-3 cells (Fig. 5, B and D). This is consistent with data obtained by Sartorelli et al. (8), demonstrating that the bHLH region is transcriptionally inactive in 3T3 cells (8). To asses the ability of the bHLH region to actively repress basal level transcription, JEG-3 and C3H10T1/2 cells were transfected with the MH100tkLUC reporter in combination with either Gal-MyoD or Gal-MyoD-bHLH. We observed that Gal-MyoD-bHLH actively repressed the basal transcription of the MH100tk-LUC reporter in C3H10T1/2 and JEG-3 cells by approximately 2- and 5-fold, respectively (Fig. 5, C and E). This set of experiments demonstrate that N-CoR directly interacts with the bHLH region of MyoD and that the bHLH region of MyoD encodes a minimal repressor domain and further supports the notion that N-CoR functions as a transcriptional corepressor of the MyoD protein. To further demonstrate that the bHLH region mediates the repressive activity in vivo, we examined the impact of deletions of the basic or HLH domain in the context of full-length MyoD linked to GAL4 to regulate the transcription of the MH100-tk-LUC reporter. Consistent with previous experiments, the bHLH region repressed transcription, in contrast to the N-terminus of MyoD (GAL4-MyoD 9N9) [Sartorelli et al. (8) demonstrated that the N-terminal region encodes an activation domain.] Interestingly, internal deletion of the basic or HLH domains in the context of full-length MyoD compromised its ability to repress the MH100-tk-LUC reporter (Fig. 5F). Interestingly TSA treatment blocked the ability of Gal-MyoD-bHLH to repress the MH100LUC reporter (Fig. 5G), consistent with a mechanism that involves the recruitment of histone deacetylases by N-CoR. MyoD Shares Common Corepressor(s) with RAR a: Expression of RAR in the Absence of Ligand Squelches the Endogenous Corepressors Retinoic acid receptor-a (RARa) in the absence of ligand binds N-CoR and represses transcription (12, 13). The RAR ligand, all-trans-RA, transforms RARa from a transcriptional repressor to a transcriptional activator. This transition is characterized by the liganddriven dissociation of N-CoR from RARa. To demonstrate that MyoD and RARa share a common corepressor in vivo (i.e. N-CoR), we performed squelching experiments. Initially, JEG-3 cells were cotransfected with the MH100tk-LUC reporter and GAL-MyoD bHLH in the presence and absence of the retinoic acid receptor (Fig. 6A). If MyoD and RARa share a common corepressor in vivo, then overexpression of RARa
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Fig. 6. MyoD Shares Common Corepressor(s) with RARa: Expression of RARa in the Absence of Ligand Squelches Repression by Endogenous Corepressors JEG-3 cells were transfected with 1 mg of the MH100-LUC reporter, 0.3 mg of either GALO or GAL-MyoD bHLH, and 2.7 mg of CMX alone or CMX-RARa in the presence or absence of all-trans-RA (1027 M). A, Overexpression of RARa blocks GAL-MyoD bHLH-mediated repression of MH100-LUC basal transcription. B, Overexpression of RARa in the presence of all-trans-RA does not inhibit GAL-MyoD bHLH-mediated repression of MH100-LUC basal transcription. Fold repression is expressed relative to the LUC activity obtained after cotransfection of the MH100-LUC reporter with GAL0 alone. Repression mediated by GAL0 is arbitarily set at 1.
should, at least in part, alleviate GAL-MyoD bHLHmediated repression of MH100tk-LUC basal transcription. We observed that overexpression of RARa (in the absence of ligand) reversed GAL-MyoD bHLH-mediated repression, suggesting that RARa and MyoD indeed share a common corepressor in vivo (Fig. 6A). In contrast, when the experiment was repeated in the presence of the RAR ligand, all-trans-RA [1027 M] (Fig. 6B), overexpression of RARa did not alleviate GALMyoD bHLH-mediated repression (Fig. 6B). Consistent with the RARa not binding with N-CoR (in the presence of ligand) and unable to squelch GAL-MyoD, bHLH mediated repression. These results were consistent with the demonstration that N-CoR binds and regulates the activity of the hierarchical transcription factor, MyoD.
DISCUSSION The regulation of muscle-specific transcription and myogenesis (i.e. muscle differentiation) is controlled by the hierarchical bHLH protein, MyoD. The cofactors, p300 and PCAF, function as critical coactivators of MyoD during the activation of the myogenic program and the induction of cell cycle arrest (8–11, 32). We have demonstrated that muscle cells express the cofactor, N-CoR, which functions as a corepressor and controls transcriptional regulation by the nuclear receptors, bHLH-LZ proteins (Mad/Mxi), the BTB/ POZ-containing oncoproteins (PLZF and LAZ3/BCL6), and RB/E2F (12, 18–24, 33). Our study also demonstrates that the cofactor, N-CoR, is repressed during myogenesis and regulates differentiation and MyoD-
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dependent transcription. These effects are mediated by the direct interaction between MyoD and N-CoR that involves the bHLH and repression regions, respectively. N-CoR expression repressed 1) the function of MyoD in the GAL4 hybrid system and 2) MyoDdependent E-box-driven expression. Furthermore, trichostatin A treatment [100,000 times more active than Na butyrate as a histone deacteylase inhibitor (34)] reversed N-CoR-dependent repression of MyoDmediated transcription and implicated a role for HDAc recruitment in corepression of MyoD activity in muscle. The Cofactor, N-CoR, Represses Myogenic Differentiation Specifically, our investigation demonstrated that the expression of the mRNA encoding the cofactor, NCoR, is repressed as proliferating myoblasts exit the cell cycle and form postmitotic myotubes. Furthermore, we observed that exogenous N-CoR expression (driven by an SV40 promoter) in myogenic cell lines inhibited morphological differentiation and resulted in lower steady state levels of MyoD mRNA and a failure to activate myogenin and p21 mRNA expression after serum withdrawal. These data demonstrate for the first time that N-CoR functions as a physiologically relevant regulator of myogenesis and provides direct evidence for the developmental role of the corepressor during mammalian differentiation. Consistent with these observations, our study showed that N-CoR could repress MyoD-mediated myogenic conversion of pluripotential 10T1/2 cells. This suggests that N-CoR functions as a corepressor of MyoD-mediated events. Puri and co-workers (9, 11) and Sartorelli et al. (8) and others (10, 32) have demonstrated that exogenous p300 and PCAF expression potentiates MyoDmediated transcription, stimulates differentiation, and promotes p21 expression and cell cycle arrest. Furthermore, the coactivators, p300 and PCAF, are abundantly expressed in differentiated muscle cells and promote MyoD-mediated conversion of pluripotent C3H10T1/2 cells (8, 11). These observations are consistent with our studies on the expression and function of the corepressor, N-CoR, in muscle and its contrasting effects on myogenesis. The opposing effects of p300/PCAF and N-CoR/Sin3B/HDAc-1 on MyoD-mediated events are a corollary to the opposing actions of the p300/CBP-PCAF-SRC [SRC-1/N-CoA-1, TIF-2/ GRIP-1, RAC-3/ACTR] coactivator complex and the N-CoR/Sin3/HDAc corepressor complex on nuclear receptor-mediated transcriptional regulation (12, 16, 17, 35, 36) and similar effects mediated by the p300/ CBP coactivator complex and the RB/histone deacetylase-corepressor complex on E2F1-mediated transcription (20, 21, 37, 38). The repression of N-CoR mRNA is also consistent with the observations that the mRNAs encoding the three orphan nuclear receptors [Rev-erbAa, RVR, and COUP-TF II] are abundantly expressed in proliferating
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myoblasts and repressed during myogenesis. Exogenous expression of these orphans has been demonstrated to antagonistically regulate myogenesis, repress MyoD mRNA expression, induce cyclin D1, abrogate the induction of myogenin and p21 mRNA after serum withdrawal, and interact with N-CoR to repress transcription (15, 25, 26, 29, 30, 39, 40). The Corepressor, N-CoR, Directly Regulates the Activity of MyoD We demonstrated that the 4RE-tkLUC MyoD-dependent reporter was specifically inhibited by N-CoR expression and that the activity of GAL-MyoD was repressed by N-CoR overexpression. Interestingly, this suggested that N-CoR functioned as a corepressor in the presence or absence of the cognate binding site for MyoD (the E-box). Our experiments suggest that one of the mechanisms involved in this process of repression was the direct interaction between MyoD and N-CoR. Additional experiments demonstrated that the interaction was mediated by the bHLH domain of MyoD and the repressor domain of N-CoR. The importance of this repressor domain-dependent interaction in the regulation of myogenesis by N-CoR was strikingly demonstrated by the observation that exogenous RIP13 expression, in absolute contrast to exogenous N-CoR expression, did not affect myogenic differentiation or cell cycle withdrawal. RIP13 is identical to N-CoR, but it lacks the N-terminal region encoding repressor domains 1 and 2, which mediate the interaction with MyoD. This highlighted the functional significance of the repressor region within N-CoR. Transfection experiments demonstrated that the bHLH region functioned as a repressor in the GAL4 hybrid system, and squelching experiments verified that the bHLH region of MyoD and the retinoic acid receptor shared a common corepressor(s), N-CoR. These observations were further reinforced by the use of the histone deacetylase inhibitor, TSA, which significantly increased the transcriptional activity of myoD and ablated N-CoR-mediated repression of MyoDdependent gene expression in myogenic cells. The bHLH Region Is a Crucial Target during Myogenesis The targeting of the bHLH region in MyoD is quite interesting in the light of several observations. First, the pocket proteins, pRb, p107, and p130, which interact with many classes of transcription factor and viral oncoproteins, participate in the induction and maintenance of the postmitotic state (41–44). Second, these pocket proteins, in their hypophosphorylated forms, specifically and directly interact with the bHLH region of the myoD gene family and mediate cell cycle withdrawal and the activation of the myogenic program (43, 44). Moreover, myoD-mediated myogenic conversion and growth arrest of fibroblastic cells is dependent on functional pRB or other pocket-like pro-
Regulation of Differentiation by N-CoR
teins (43). Third, a number of groups have recently demonstrated that the silencing of E2F-dependent gene expression involves a complex between E2F, hypophophorylated RB protein, and the histone deacetylases (20, 21, 38). It has been suggested that active transcriptional repression by hypophosphorylated RB involves a modification of chromatin structure, which leads to the repression of E2F-dependent gene expression and cellular proliferation. Control of myoblast differentiation is also regulated by factors that effect the phosphorylation of the RB. RB activity is controlled by cell division kinase (cdk) complexes with the cyclins (reviewed in Ref. 45). The cyclins and cdks inhibit muscle-specific transcription via the hyperphosphorylation of RB, which leads to the activation of E2F1 and cellular proliferation (46, 47). The activity of cdks is regulated at the level of synthesis of the subunit partners (e.g. cyclins) of the complex and by binding of inhibitors (e.g. p21Cip1/Waf1) (reviewed in Ref. 45). The critical role of these cell cycle regulators in myogenesis has been demonstrated by 1) the inhibition of myogenesis by forced expression of cyclin D1 by mechanisms dependent/and independent of pRB hyperphosphorylation and MyoD phosphorylation (46– 48) and 2) the ectopic expression of p21 in growing myoblasts results in cell cycle arrest (4, 6, 49). We suggest that, during cell cycle exit and the activation of the myogenic program, the multiprotein corepressor complex that functions to repress the activity of MyoD begins to play a critical role in mediating the effects of RB on E2F-dependent gene expression and the inhibition of cell cycle-related transcription. Coactivators and Corepressors, Critical Regulators of Cellular Proliferation and Myogenic Differentiation This study contributes to an emerging and complex story about the crucial role of cofactors in myogenesis.
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Whether the domains involved and the subsets of cofactors used during myogenesis display specificity with respect to the 1) class of transcription factor involved, 2) target gene(s), and 3) cell cycle remains to be resolved. The evidence to date supports the notion that the coactivator and corepressor complexes function in both the proliferative phases and postmitotic states of myogenesis. It has been observed that the coactivator complex p300 mediates E2F-dependent S-phase gene expression, DNA synthesis, and cell cycle progression (37). This process is dependent and mediated by the phosphorylation of RB or the other pocket proteins by the cyclin/cdk pathway. During withdrawal from the cell cycle and myogenic differentiation, a number of key events take place: 1) pRB becomes hypophosphorylated and recruits histone deacetylase (in a pocket domain-dependent fashion) to directly repress E2F-dependent gene expression (20, 21, 38); 2) the hypophosphorylated pRB and MyoD directly bind to each other through an interface that involves the pocket and the bHLH regions, consistent with the effects of MyoD on the myogenic pathway and cell cycle withdrawal, respectively (43); and 3) concomitant with the above events, p300 and PCAF directly interact with MyoD and potentiate myogenic differentiation and permanent withdrawal from the cell cycle (8–11, 32). Our study demonstrates that the N-CoR corepressor complex can prevent activation of p21 and myogenesis; moreover, it can directly mediate the repression of MyoD function (see Fig. 7). Clearly, the E2F- and myogenic-specific bHLH proteins have opposing functions during proliferation and differentiation. Furthermore, this and other studies suggest that the coactivator and corepressor complexes have crucial roles, both in division and differentiation. The function of E2F and MyoD is dictated by a dynamic balance of cofactor interactions, involving either corepressor and coactivator complexes, that
Fig. 7. Schematic Diagram Depicting the Functional Role of N-CoR in the Regulation of Myogenesis and Cell Cycle Withdrawal The functional role of the coactivators, p300 and PCAF, in this model is supported by the work of Sartorelli et al. (8), Puri et al. (9, 11), and Eckner et al. (10).
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lead to histone deacetylation or acetylation, respectively, which influence nucleosome condensation in a targeted/localized fashion in the vicinity of gene subsets specifically involved in either cell division or differentiation, respectively. Ultimately, these events are controlled or triggered by external stimuli (e.g. growth factors, hormones, etc). Conclusion We propose that this study provides a paradigm for corepressor function in other systems of differentiation and highlights that the regulation of mammalian differentiation by hierarchical classes of transcription factors involves a fine balance between coactivator and corepressor complexes that are tightly linked to the regulation of the cell cycle.
MATERIALS AND METHODS Cell Culture and Transient Transfections 10T1/2, NIH3T3 fibroblasts, or JEG-3 (human choriocarcinoma) cells were cultured for 24 h in DMEM supplemented with 10% FCS in 6% CO2 before transfection. Mouse myogenic C2C12 cells were grown in DMEM supplemented with 20% FCS in 6% CO2. Before transfection 10T1/2, JEG-3, or C2C12 cells were cultured in DMEM containing 5% charcoalstripped FCS in 6% CO2 at 37 C. Cells grown in six-well dishes to 60–70% confluence were transiently transfected with 1 mg of the G5E1bLUC or MH100 LUC reporter plasmid and 1–3 mg of a mixture of GAL4-MyoD chimera in the presence and absence of the specified cofactor (N-CoR, Sin3B, and HDAc-1) by the DOTAP/DOSPER (Boehringer Mannheim, Indianapolis, IN)-mediated procedure as described previously (15). Transfection with the 4RE-tkLUC used 1 mg of the reporter, cotransfected with 1–3 mg of the specified cofactor. The DNA/DOTAP/DOSPER mixture was added to the cells in 3 ml of fresh medium, and 24 h after transfection the medium was replaced. Cells were harvested and assayed for luciferase activity as previously described after an additional 24 h. Cells were also transfected in 12-well plates with approximately one third of the volumes and quantities described above. Each transfection experiment was performed at least three times to overcome the variability inherent in transfections. Luciferase Assays Luciferase activity was assayed using a Luclite kit (Packard Instruments, Meriden, CT) according to the manufacturer’s instructions. Briefly, cells were washed once in PBS and resuspended in 150 ml of phenol red-free DMEM and 150 ml of Luclite substrate buffer. Cell lysates were transferred to a 96-well plate, and relative luciferase units were measured for 5 sec in a Wallac Trilux 1450 microbeta luminometer. Construction of Stable Cell Lines C2C12 cells were stably transfected at approximately 40% confluence using the DOTAP (Boehringer Mannheim)-mediated procedure as described previously (29). Briefly, a 1 ml DNA/DOTAP mixture (containing 20 mg of pSG5-N-CoR or RIP13, 1.5 mg of pCMV-NEO, 75 ml of DOTAP, 75 ml of DOSPER in 20 mM HEPES, 150 mM NaCl, pH 7.4) was added
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to the cells in a 150-mm dish and 20 ml of fresh culture medium. The cells were then grown for a further 24 h to allow cell recovery and for high-level pCMV-NEO expression before selection. Stable transfectants were isolated after 7–14 days selection in DMEM supplemented with 20% FCS and 400 mg/ml G418. C3H10T1/2 Cell Myogenic Conversion To determine whether pSG5-NCoR was capable of repressing the SV40 promoter 0.3 mg, 1 mg, and 3 mg of either pSG5 or pSG5-N-CoR were cotransfetced with the pGL-3-Control/ SV40-LUC reporter in C3H10T1/2. For the myogenic conversion assay, C3H10T1/2 fibroblasts were grown to 60% confluence in 12-well dishes. Cells in each well were transfected by the DOTAP/DOSPER (Boehringer Mannheim)-mediated procedure, using 1 mg of pSG5-MyoD in combination with either 1 mg of pSG5-N-CoR or pSG5 carrier DNA to total 2 mg per transfection. Twenty four hours after transfection, fresh medium, 10% FCS in DMEM, was added until 100% confluent, after which the media was changed to DMEM plus 2% horse serum. Cells were grown under these conditions for 6 days, with media changes occurring every 2 days. Immunostaining was then performed using a monoclonal antibody directed toward the fast isoform of the major thick filament protein, skeletal myosin heavy chain (Sigma Chemical Co., St. Louis, MO; clone MY32). This procedure is described in detail elsewhere (8). GST Pulldowns GST and GST fusion proteins were expressed in Escherichia coli (BL21) and purified using glutathione-agarose affinity chromatography as described previously (55). The GST fusion proteins were analyzed on 10% SDS-PAGE gels for integrity and to normalize the amount of each protein. The Promega Corp. (Madison, WI) TNT-coupled transcription-translation system was used to produce [35S]methionine-labeled MyoD and N-CoR proteins that were visualized by SDS-PAGE. In vitro binding assays were performed with glutathione agarose beads (Sigma Chemical Co., St. Louis, MO) coated with approximately 500 ng GST fusion protein and 2–30 ml [35S]methionine-labeled protein in 200 ml of binding buffer containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonident P-40, 5 mg of ethidium bromide, and 100 mg of BSA. The reaction was allowed to proceed for 1–2 h at room temperature with rocking. The affinity beads were then collected by centrifugation and washed five times with 1 ml of binding buffer without the ethidium bromide or BSA. Beads were resuspended in 20 ml of SDS-PAGE sample buffer and boiled for 5 min. The denatured proteins were run on a 10% SDS-PAGE gel that was subsequently treated with Amersham Amplify fluor (Amersham, Arlington Heights, IL), dried, and autoradiographed. Plasmids GAL-MyoD, GAL-MyoD N-terminal, GAL-MyoD DHLH, GAL-MyoD Dbasic, GAL-MyoDbHLH, 4REtk-LUC, G5E1b-LUC, MH100-LUC, pSG5-N-CoR, and CMX-RAR are described elsewhere (8, 17). pSG5-MyoD was constructed by excising MyoD cDNA from the pEMSVscribe (Moloney Sarcoma Virus)-based expression vector pEMSV-MyoD and recloning into the EcoRI site of the pSG5 vector. RNA Extraction, Northern Hybridization, and Probe Preparation Total RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method. Northern blots, random priming, and hybridizations were performed as described previously (56). The actin probes used have been described by Bains et al. (57). The mouse myogenin (58) and MyoD (59) cDNAs were excised from the pEMSVscribe (Moloney Sarcoma Virus)-based expression vectors. Mouse Cyclin D1 was excised from pGEX-3X-CYL1 (60), and mouse p21 was excised from pCMW35, an unpublished clone encoding mouse p21 from the Vogelstein laboratory. HDAC1 was excised from
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pBluescript-HDAC1 using SalI restriction digest. mSin3BLF was excised from pcDNA3.1-mSin3BLF with HindIII. cDNA probes were radioactively labeled by random priming. DNA fragment (50–100 ng) was boiled with 20 ng random primers (pdN6; Pharmacia Biotech, Piscataway, NJ). The DNA was then incubated at room temperature overnight with RPB (50 mM Tris, pH 7.5, 10 mM MgCl2, 200 mM dATP, dGTP, dTTP), 10 ml [a-32P]-dCTP (Bresatec Ltd., Thebarton, Australia), and 2 U of Klenow polymerase. Probes were purified using a NICK column (Pharmacia Biotech) according to the manufacturer’s instructions. Quantitation of 18S rRNA was performed using the following 25-mer oligonucleotide, 59CATGGTAGGCACGGCGACTACCATC-39. To label the 18S rRNA oligonucleotide probe, 100 ng of the 25-mer were incubated at 37 C for 2 h with 1 ml polynucleotide kinase (PNK) (Boehringer Mannheim), 2 ml 103 PNK buffer (Boehringer Mannheim), and 3 ml 32P-g dATP in a total volume of 20 ml. The probe was purified on a NAP column (Pharmacia Biotech) as directed by the manufacturers.
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12. 13.
14.
15.
Acknowledgments Received January 11, 1999. Revision received February 25, 1999. Accepted March 11, 1999. Address requests for reprints to: Dr. George E. O. Muscat, Centre for Molecular & Cellular Biology, University of Queensland, Research Road Ritchie Research Laboratories, B402A St Lucia, Brisbane, Queensland, Australia 4072. E-mail:
[email protected].
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