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

项目摘要 p27是限制正常细胞增殖的CDK抑制剂。我们发现p27 C-末端磷酸化在T157 AKT增加了T198和p27pT157pT198（p27pTpT）与新蛋白的相互作用 伙伴我们最近发现C-末端p27 pTpT磷酸化促进了其与cJun的结合。 基因组分析显示，p27与cJun共募集到超过一半的cJun染色质结合位点， 激活或抑制靶基因。p27/cJun激活的靶标包括TGFB2，并且与EMT相关，并且 上调干细胞并改变细胞粘附和迁移的程序。抑制的靶基因谱 p27/cJun的表达提示p27 pTpT对抗组织分化。p27/cJun靶基因的一个子集结合到一个 STAT3共有基序和p27、cJun和STAT3似乎都结合并共调节cMYC。值得注意的是， p27 pTpT是干细胞效能的驱动因素：细胞p27 pTpT上调球体、SOX 2、NANOG和cMYC 和体内肿瘤起始细胞。我们的TG-p27 CK-DD小鼠转基因的p27磷酸化模拟突变体， 未能结合细胞周期蛋白/CDKs，显示多器官过度生长和体积增加，表明p27 CK-DD 消除了WTp 27抑制正常茎和祖细胞扩增的作用。这项补助金调查的作用 p27在正常细胞和发育中作为转录调节因子。我们的数据支持这样的假设， WTp 27在分化过程中发挥重要的转录作用，以限制干细胞和祖细胞的扩增。 各种组织。相反，在C-末端磷酸化后，p27与cJun、STAT3和其他因子相互作用， 以扩增或维持组织干细胞或祖细胞并对抗分化。我们将比较p27WT， AIM1中的p27 CDK和p27 CK-DD MEFS，以评估p27如何与染色质上的cJun和STAT 3相互作用， 控制从G0到G1中期和G1/S期过渡的细胞周期中的基因表达。我们将 研究增加的C-末端p27磷酸化是否改变共调节因子和靶基因选择， 消除了p27在静止细胞中的共抑制功能。在AIM 2中，我们将主动转录 从转录失活的异染色质中分离常染色质，并进行ChIP-seq和ChIP-Mass Spec， 鉴定涉及反式激活因子与阻遏因子复合物的染色质相关p27相互作用组。AIM3 将研究小鼠发育，组织分化，成年干细胞和祖细胞群体在TG-p27 CK- DD和TG-p27 CK-小鼠。我们将测试p27 CK-DD是否赋予MEFs更强的重编程潜力， 破坏iPSC向胚状体的分化。这项工作将阐明p27作为一个新的功能， 具有组织干细胞控制和潜能意义的磷酸化依赖性转录调节因子 在再生医学中的应用p27或本文鉴定的p27靶基因的受控调节， 可能提供扩张或再生血液和其他组织的潜力。了解p27/cJun 调节组织发育的转录程序可能会发现新的治疗靶点， 组织再生并阐明分化和生长控制界面上的其他人类疾病。

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.