Novel roles for p27 as transcriptional co-regulator of cJun in stem cells and development

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Project Summary p27 is a CDK inhibitor that limits normal cell proliferation. We showed p27 C-terminal phosphorylation at T157 and T198 by AKT increases in mid G1 and p27pT157pT198 (p27pTpT) facilitates interaction with novel protein partners. We recently showed C-terminal p27pTpT phosphorylation promotes its association with cJun. Genomic profiling showed p27 is co-recruited with cJun to over half of cJun chromatin binding sites to either activate or repress target genes. p27/cJun activated targets include TGFB2, and are associated with EMT, and programs that upregulate stem cells and alter cell adhesion and migration. Profiles of target genes repressed by p27/cJun suggest that p27pTpT opposes tissue differentiation. A subset of p27/cJun target genes bind to a STAT3 consensus motif and p27, cJun and STAT3 all appear to bind and co-regulate cMYC. Notably, p27pTpT is a driver of stem cell potency: cellular p27pTpT upregulates spheres, SOX2, NANOG and cMYC and tumor initiating cells in vivo. Our TG-p27CK-DD mice transgenic for a p27 phosphomimetic mutant that fails to bind cyclin/CDKs, show multi-organ overgrowth and increased size, suggesting that p27CK-DD abrogates WTp27 actions to restrain normal stem and progenitor expansion. This grant investigates the role of p27 as a transcriptional regulator in normal cells and development. Our data support the hypothesis that WTp27 plays important transcriptional roles during differentiation, to limit stem and progenitor cell expansion in various tissues. In contrast, upon C-terminal phosphorylation, p27 interacts with cJun, STAT3 and other factors to expand or maintain tissue stem or progenitor cells and oppose differentiation. We will compare p27WT, p27CDK and p27CK-DD MEFS in AIM1 to evaluate how p27 interacts with cJun and STAT3 on chromatin to govern gene expression across the cell cycle from G0, to mid-G1 and the G1/S phase transition. We will investigate if increased C-terminal p27 phosphorylation alters co-regulator and target gene selection and abrogates the co-repressive functions of p27 in quiescent cells. In AIM2, we will separate actively transcribed euchromatin from transcriptionally inactive heterochromatin and carry out ChIP-seq and ChIP-Mass Spec to identify the chromatin associated p27 interactome involved in transactivator versus repressor complexes. AIM3 will study mouse development, tissue differentiation, and adult stem and progenitor populations in TG-p27CK- DD and TG-p27CK- mice. We will test if p27CK-DD confers stronger reprogramming potential on MEFs and disrupts differentiation of iPSC into embryoid bodies. This work will elucidate a novel function of p27 as a phosphorylation-dependent transcriptional regulator with implications for tissue stem cell control and potential applications in regenerative medicine. Controlled modulation of p27, or of p27 target genes identified herein, might offer potential to expand or regenerate hematologic and other tissues. Understanding how p27/cJun transcriptional programs regulate tissue development might identify new therapeutic targets to be exploited for tissue regeneration and illuminate other human diseases at the interface of differentiation and growth control.