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 replicationprotein PCNA, p21WAF1/CIP1 is a multifunctional protein that interacts with many cellularbinding partners, including a number of transcriptional regulators. Previously, wecharacterised p21 derepression of the p300 sumoylation-dependent transcriptionalrepression domain, CRD1. Such repression domains are at least partially dependent uponrecruitment of histone deacetylase (HDAC) complexes but the mechanism through whichp21 selectively disrupts CRD1 activity remains unknown. Here, we demonstrate thatdistinct motifs in the C-terminus of p21 are required for regulation of p300 CRD1function and that this effect does not correlate with Cyclin or PCNA binding. Throughthe creation of N-terminal glutathione-s-transferase fusion proteins, which also overcomethe problems of instability that result from many p21 mutations, we investigated p21binding to HDACs. Although p21 binds both Class I and Class II HDACs in vitro, onlyweak association with HDAC1 and 2 is seen in cells. Mutation of the p21 PCNA bindingdomain significantly increases this interaction suggesting that binding is mutuallyexclusive and only naturally occurs under certain conditions. Binding of HDACs alsofailed to correlate with CRD1 inducibility, suggesting that p21 targets othertranscriptional repression complexes to mediate this effect.