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

项目摘要 P27是一种CDK抑制因子，可以限制正常细胞的增殖。我们在T157显示了p27C-末端的磷酸化 AKT导致G1期中期T198增加，p27pT157pT198(P27pTpT)促进与新蛋白的相互作用 合伙人。我们最近发现，C端p27pTpT的磷酸化促进了它与cJun的结合。 基因组图谱显示，p27与cjun共招募到超过一半的cjun染色质结合位点 激活或抑制靶基因。P27/cJun激活的靶点包括TGFB2，并与EMT相关，以及 上调干细胞和改变细胞黏附和迁移的程序。抑制靶基因的表达谱 P27/cJun提示p27pTpT反对组织分化。P27/cJun靶基因的一个子集与一个 STAT3共识基序和p27、cJun和STAT3似乎都结合并共同调节cMYC。值得注意的是， P27pTpT是干细胞潜能的驱动力：细胞p27pTpT上调球体、SOX2、NANOG和cMYC 体内的肿瘤启动细胞。我们的TG-p27CK-DD小鼠转基因p27模拟磷酸化突变体 未能与细胞周期蛋白/CDK结合，表现为多器官过度生长和体积增大，提示p27CK-DD 取消WTp27抑制正常茎和祖细胞扩张的作用。这项拨款调查了 P27在正常细胞和发育过程中作为转录调控因子。我们的数据支持这样一种假设 WTp27在分化过程中发挥重要的转录作用，限制干细胞和祖细胞的增殖。 各种纸巾。相反，在C末端磷酸化后，p27与cJun、STAT3和其他因子相互作用 扩增或维持组织干细胞或祖细胞并反对分化我们将比较p27WT， P27CDK和p27CK-DD MEFS，以评估p27如何与cJun和STAT3在染色质上相互作用 控制细胞周期从G0到G1期中期和G1/S期的基因表达。我们会 研究C-末端p27磷酸化增加是否改变共调控基因和靶基因选择以及 在静止细胞中取消p27的共抑制功能。在AIM2中，我们将分开积极转录 从转录不活跃的异染色质中提取常染色质，并进行CHIP-SEQ和CHIP-MS 确定参与反式激活物与抑制物复合体的染色质相关p27相互作用体。AIM3 将在Tg-p27CK中研究小鼠的发育、组织分化以及成体干细胞和祖细胞种群。 DD和TG-p27CK-小鼠。我们将测试p27CK-DD是否赋予MEF更强的重新编程潜力和 破坏IPSC向类胚体的分化。这项工作将阐明p27作为一种新的 磷酸化依赖的转录调控因子及其对组织干细胞控制和潜能的影响 在再生医学中的应用。P27或这里确定的p27靶基因的受控调制， 可能提供扩大或再生血液和其他组织的潜力。了解p27/cJun如何 调控组织发育的转录程序可能会确定新的治疗靶点 在分化和生长控制的界面上，组织再生和照亮其他人类疾病。

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.