Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE HARTERLABORATORYPOSTDOCTORAL FELLOWMrinal Ghosh, Ph.D.COLLABORATORSJane Flint, Ph.D. 1Saikh Jaharul Haque, Ph.D. 2Tony Hunter, Ph.D. 31Princeton Univ., Princeton, NJ2Dept. of Cancer Biology, CCF3Salk Institute, La Jolla, CAThe Department of Molecular BiologyAdenovirus Holds Keys to DissectingMechanisms of Cell Cycle, ProliferationThe human adenovirus relies largely on themammalian host DNA synthesis apparatusfor its replication in quiescent or terminallydifferentiated cells. Consequently, this virus hasevolved a specific gene (E1A) to induce cellularDNA synthesis in these cells. E1A, however, cancarry on this activity outside the context of thevirus, and in fact, promote DNA synthesis in cellsblocked in G1, either by TGF-β signaling, the useof a ras neutralizing antibody, or a DNAdamaging agent.Our primary focus isto identify the mechanismsby which E1A affects cellcyclepathways and, in turn,understand some of themolecular details underlyingcell proliferation andterminal differentiation.E1A is best knownfor targeting the retinoblastomafamily of proteins(pRb, p107, p130), whichare co-repressors oftranscription. We recentlydiscovered that E1A couldalso inactivate the cyclindependentkinase (cdk)inhibitors p27 and p21.These interactions respectivelypromote TGF-βtreatedcells into S phase andlead to the induction ofDNA synthesis in differentiatedmuscle cells. In effect, these studiesestablished an entirely new way in which E1Acould modulate the cell cycle.We have also discovered that E1A canperturb the mechanistic links between histoneMal, A., Poon, R. Y. C., Howe, P.P., Toyoshima, H., Hunter, T., and M.L. Harter(1996) The E1A oncoprotein disables the CDK inhibitor p27Kip1 in TGF-ß treated cells.Nature 380:262-265.Mal, A., Chattopadhyay, D., Ghosh, M.K., Poon, R.Y. Hunter, T., and M.L. Harter(2000) p21 and retinoblastoma protein control the absence of DNA replication in terminallydifferentiated muscle cells. J. Cell Biol. 149:281-292.Mal, A., Sturniolo M., Schiltz, R.L., Ghosh, M.K., and M.L. Harter (2001) A role for histonedeacetylase HDAC1 in modulating the transcriptional activity of MyoD: Inhibitionof the myogenic program. EMBO J. 20:1739-1753.Chattopadhyay, D., Ghosh, M.K., Mal, A., and M.L. Harter (2001) Inactivation of p21by E1A leads to the induction of apoptosis in DNA-damaged cells. J. Virol. 75:9844-9856.Mal, A., and M. L. Harter (2003) MyoD is functionally linked to the silencing of amuscle-specific regulatory gene prior to skeletal myogenesis. Proc. Natl. Acad. Sci.USA 100:1735-1739.Marian L.(Nikki) Harter, Ph.D.modifications and chromatin function toreactivate E2F-responsive genes that arenecessary for inducing DNA synthesis in quiescentcells. E1A has the ability to target chromatin andthereby interact with a promoter-bound p130,which associates with the histone methylaseSUV39H1. This interaction induces p130’sremoval from the E2F-regulated promoters, andconsequently, this causes the surroundingnucleosomal histones to lose their methylationand become alternativelymodified (phospho-acetylated),an event that leads to theactivation of transcription.These studies provide the firstexample of a viral protein thatcan specifically affect chromatinfunction.The molecularmechanism(s) that are responsiblefor suppressing MyoD’stranscriptional activities inundifferentiated skeletal musclecells have not yet been determined.We have now shownthat MyoD associates with ahistone deacetylase-1 (HDAC1)in these cells and that thisinteraction is responsible forsilencing MyoD-dependenttranscription of muscle specificgenes. We have also shown thatan endogenous MyoD can beacetylated by P/CAF but onlywhen cells have been induced to differentiate.These results provide for a model, whichpostulates that MyoD may be codependent onHDAC1 and P/CAF for temporally controlling itstranscriptional activity before and after thedifferentiation of muscle cells.Most of the genes (e.g., myogenin) that arecentral to the process of skeletal muscledifferentiation remain in a transcriptionally silentor “off ” state until myoblasts are induced todifferentiate. Importantly, we have now shownthat both MyoD and HDAC1 occupy themyogenin promoter in myoblasts, which issurrounded by methylated H3 histones. Aftermyoblasts are induced to differentiate, however,HDAC1 is no longer detected at this promoter,and instead, both MyoD and acetyltransferase P/CAF now occupy this promoter. In addition,enrichment of histone H3 acetylation andphosphorylation is now observed at this promoter.These data suggest that in addition tobeing an activator of differentiation-specificgenes, MyoD can also act as a transcriptionalrepressor in proliferating myoblasts while inpartnership with a histone deacetylase.106