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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 7, 2002 as doi:10.1096/fj.02-0123fje. |
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-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor Myogenin 1
,2
,*,,*3
Istituto di Tecnologie Biomediche, CNR, 00137 Rome, Italy;
* Cell Metabolism and Pharmacokinetics Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy; and
Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy
3Correspondence: Istituto di Tecnologie Biomediche, CNR, Viale Marx 43, 00137 Rome, Italy. E-mail: passananti{at}itbm.rm.cnr.it
SPECIFIC AIMS
RPB3 is a RNA polymerase II (pol II) 
-like core subunit. Here we show that RPB3 directly contacts the myogenic transcription factor Myogenin, mediating in this way Myogenin/pol II interaction. RPB3 contacts only Myogenin; it does not bind the other myogenic factors such as MyoD, Myf5, and MRF4. These findings coincide with the Myogenin role played principally in differentiation/maintenance of the myogenic phenotype rather than in muscle determination. The interaction between RPB3 and Myogenin suggests a direct link between a tissue-specific transcription factor and pol II and appears to be critical for the ‘active’ maintenance of muscle differentiation state.
PRINCIPAL FINDINGS
1. RPB3 interacts with Myogenin
To identify protein/s interacting with RPB3, yeast two-hybrid experiments were performed using a cDNA library prepared from human skeletal muscle. Of 30 positive clones analyzed, two independent clones encoded for the basic helix loop helix (bHLH) myogenic transcription factor Myogenin. To map the regions of RPB3 and Myogenin directly involved in the interaction, RPB3 was divided into four portions. A region that spans amino acids 74–138 named Sud represents the minimal RPB3 region required to contact Myogenin. We demonstrated that Sud efficiently competes with RPB3/Myogenin interaction in pull-down competition experiments, suggesting that Sud can act as a dominant negative molecule. Similarly, the Myogenin region directly involved in RPB3 contact was identified using a series of four Myogenin deletions. The HLH domain of Myogenin alone retained the ability to contact RPB3. In summary, these data indicate that RPB3-Sud fragment mediates the interaction with the Myogenin HLH domain.
2. RPB3 connects Myogenin to pol II
To answer the question of whether Myogenin contacts RPB3 in isolation or as part of the pol II enzyme, we performed the series of immunoprecipitation experiments presented in Fig. 1
. Co-immunoprecipitation experiments performed in differentiated C2C7 myogenic cells using a polyclonal antibody against the largest subunit of pol II (RPB1) clearly revealed the presence of Myogenin in the immunocomplex (Fig. 1A
). A similar result was obtained performing the reciprocal experiment, using Myogenin antibody in immunoprecipitation and pol II antibody in Western blot analysis of the immunoprecipitated complexes (Fig. 1B
). Next we looked for evidence that RPB3 is the molecule responsible for linking Myogenin to pol II; therefore, Sud was used as a dominant negative molecule. In fact, missing the portion of RPB3 carrying the pol II -like signature, Sud appears not to affect significantly pol II assembly. On the other hand, Sud contains RPB3 regions able to bind Myogenin so that it can interfere with RPB3/Myogenin contact. Overexpression of Myogenin with increasing amounts of Sud in C3H10T1/2 fibroblasts indicated that Sud efficiently removed Myogenin from pol II (Fig. 1C
). These results indicate that RPB3 is able to contact Myogenin when it is part of pol II core.
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3. RPB3 expression is modulated during murine C2C7 myoblast differentiation
We analyzed RPB3 protein level during the onset of differentiation of murine C2C7 cells. We demonstrated that RPB3 level is low in cycling C2C7 cells, but increases of 
twofold in high-density orientated cells were concomitant with the beginning of the differentiation program. Levels of the RPB1 and RPB11 pol II subunits did not show any appreciable variation during C2C7 differentiation.
4. RPB3/Myogenin interaction affects myogenic differentiation
The observations that RPB3 contacts Myogenin and is regulated during muscle differentiation led us to consider that this subunit is directly involved in Myogenin-dependent transcription. To test this hypothesis RPB3 was cotransfected with Myogenin into C3H10T1/2 fibroblasts and transcription was determined by measuring reporter gene activity driven by both the artificial four E-box DNA binding sites (4RE), and the naturally occurring muscle creatine kinase enhancer element. Overexpression of RPB3 slightly stimulated Myogenin-dependent transcription from both enhancers. Moreover, the same cotransfection experiments were performed using Sud as a dominant negative construct. Analysis of the results clearly demonstrates that expression of Sud markedly inhibited Myogenin-dependent transcription. These results show that RPB3 plays a specific role in Myogenin-dependent transcription. To test the role of the RPB3/Myogenin interaction in myogenic differentiation, we used the myogenic conversion assay because one of the myogenic bHLH cDNA is transfected into C3H10T1/2 fibroblasts, which subsequently undergo myogenesis (Fig. 2
A). Cotransfection of RPB3 with Myogenin slightly increased the percentage of myogenic cells resulting from Myogenin transfection, whereas cotransfection of Sud with Myogenin significantly inhibited cell differentiation. This effect of Sud on Myogenin in C3H10T1/2 fibroblasts was further confirmed when transfected cells were analyzed by Western blot. As shown in Fig. 2B
, concomitant with Sud expression, the protein level of the terminal differentiation marker myosin heavy chain (‘MHC’) specifically decreased. Moreover, Sud inhibitory effect was directly visualized by immunofluorescence experiments (Fig. 2C
, bottom panel), where cells that are positive for Sud (green) are negative for MHC expression (red). This mutual exclusion expression was not observed when Myogenin was cotransfected with empty vector pIRES-EGFP (Fig. 2C
, top panel). These observations confirmed the evidence that RPB3 plays a crucial role in muscle differentiation.
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CONCLUSIONS AND SIGNIFICANCE
RPB3 is an essential component of the transcriptional machinery. Besides its well-documented structural function in the pol II subassembly, a role for RPB3 in transcriptional regulation is emerging. In fact, despite being essential for cellular viability and being detected in all adult tissues analyzed, RPB3 protein exhibits a modulated pattern of expression with clear peaks in heart and muscle tissues. Recently, the oncoprotein EWS has been found to modulate pol II activity by interacting with two pol II subunits, RPB3 and RPB5. In yeast, one selected RPB3 mutant exhibits a pronounced defect in activator-dependent transcription, supporting the idea that RPB3 inside pol II may be a target for transcriptional activators. We have previously demonstrated that doxorubicin down-regulates expression of RPB3. This drug inhibits myogenic differentiation of C2 myoblasts by down-regulating MyoD and Myogenin. On the basis of these observations, we isolated proteins that interact with RPB3 in muscle. Here, we describe a novel specific interaction between RPB3 and Myogenin. The portion of RPB3 protein named Sud is necessary to contact Myogenin. Sud contains a cysteine-rich region that resembles a potential zinc finger motif. This region is conserved only in eukaryotic pol II and it is not present in the corresponding subunits of pol I and pol III; thus, Sud can be involved in function(s) specific for pol II. The HLH domain of Myogenin is engaged in contacting the cysteine-rich region of RPB3. This is consistent with the finding that the three zinc fingers of the transcription factor Sp1 are necessary and sufficient for binding the HLH domain of Myogenin.
Although the HLH domain is highly conserved among the family of myogenic bHLH proteins, we found that RPB3 is able to contact only Myogenin. This finding agrees well with the role of Myogenin, which is principally involved in the differentiation/maintenance of the myogenic phenotype rather than in muscle determination like MyoD and Myf5. Disagreement exists with respect to the copy number of RPB3 in eukaryotic pol II, suggesting that this protein may play a regulatory function by varying its stoichiometry with other RNA pol II subunits and/or core promoter elements. Although the structure of yeast pol II has been described and the stoichiometry of yeast RPB3 has now been established, the results presented in this paper support the hypothesis that in higher eukaryotes during phenomena like tissue differentiation, pol II may partially change its composition. We analyzed RPB3 transcriptional effects testing several promoters by luciferase assay. RPB3 exerted a modest positive effect limited to Myogenin-mediated trans-activation. We speculate that RPB3 cannot have by itself a strong stimulatory effect on transcription since it plays its role as part of the core of pol II. We consider that overexpression of a single subunit could only partially affect pol II activity (Fig. 3
). The importance of RPB3/Myogenin interaction inside pol II in Myogenin-dependent transcription and muscle differentiation was more clearly demonstrated by overexpressing the Sud portion of RPB3 as a dominant negative molecule. Sud, which misses both the -like pol II-signatures, cannot interfere with the role of RPB3 in the assembly of pol II. On the other hand, Sud competes with RPB3 for the binding to Myogenin, displacing this last molecule from pol II holoenzyme complex. Thus, the results obtained from overexpressing Sud can be ascribed principally to the failure of the interaction pol II-RPB3/Myogenin. In conclusion, the data presented here support the notion that RPB3, in addition to the well-described structural function, is involved in Myogenin-dependent activation of transcription during muscle differentiation.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0123fje; to cite this article, use FASEB J. (August 7, 2002) 10.1096/fj.02-0123fje 
2 These authors equally contributed to this work.
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