title: p21WAF1/CIP1 regulates the p300 sumoylation motif CRD1 through a C-terminal domain independently of cyclin/CDK binding.
abstract: Although best known for its ability to inhibit Cyclin/Cdk complexes and the replication protein PCNA, p21(WAF1/CIP1) is a multifunctional protein that interacts with many cellular binding partners, including a number of transcriptional regulators. Previously, we characterized p21 derepression of the p300 sumoylation-dependent transcriptional repression domain, CRD1. Such repression domains are at least partially dependent upon recruitment of histone deacetylase (HDAC) complexes but the mechanism through which p21 selectively disrupts CRD1 activity remains unknown. Here, we demonstrate that distinct motifs in the C-terminus of p21 are required for regulation of p300 CRD1 function and that this effect does not correlate with Cyclin or PCNA binding. Through the creation of N-terminal glutathione-s-transferase fusion proteins, which also overcome the problems of instability that result from many p21 mutations, we investigated p21 binding to HDACs. Although p21 binds both Class I and Class II HDACs in vitro, only weak association with HDAC1 and 2 is seen in cells. Mutation of the p21 PCNA binding domain significantly increases this interaction suggesting that binding is mutually exclusive and only naturally occurs under certain conditions. Binding of HDACs also failed to correlate with CRD1 inducibility, suggesting that p21 targets other transcriptional repression complexes to mediate this effect.
authors.affiliation: School of Life Sciences, Division of Gene Regulation and Expression, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: Elisa
authors.initials: E
authors.lastname: Garcia-Wilson
authors.forename: Neil D
authors.initials: ND
authors.lastname: Perkins
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002453:Cell Cycle; D002460:Cell Line; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism / Q000502:physiology*; D018844:Cyclin-Dependent Kinases / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D014162:Transfection; D050880:p300-CBP Transcription Factors / Q000378:metabolism*
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D018076:DNA, Complementary; D050880:p300-CBP Transcription Factors; D005982:Glutathione Transferase; D018844:Cyclin-Dependent Kinases; D006655:Histone Deacetylases