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

p27 作为 cJun 转录共调节因子在干细胞和发育中的新作用

• 批准号: 10031373
• 负责人: JOYCE MARIE SLINGERLAND
• 金额: $ 59.52万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10031373
• 关键词: Adult; Binding; Binding Sites; Biological Assay; Bone Marrow Stem Cell Transplantation; Breast Epithelial Cells; C-terminal; CCNE1 gene; CDK2 gene; CDK4 gene; Cell Adhesion; Cell Cycle; Cell Cycle Arrest; Cell Differentiation process; Cell Proliferation; Cells; ChIP-seq; Chromatin; Complex; Consensus; Cyclin D1; Cyclins; Data; Defect; Development; Differentiation and Growth; Disease; Euchromatin; Exhibits; G1 Phase; Gene Expression; Genes; Genetic Transcription; Gigantism; Grant; Hematology; Hematopoietic; Heterochromatin; Hyperplasia; Knock-out; Mediating; Modeling; Mus; Mutation; Natural regeneration; Normal Cell; Normal tissue morphology; Periodicity; Phase Transition; Phenotype; Phosphorylation; Play; Population; Proteins; Proto-Oncogene Proteins c-akt; Regenerative Medicine; Repressor Proteins; Role; S Phase; STAT3 gene; Signal Transduction; Site; Stem Cell Development; TGFB2 gene; Testing; Therapeutic; Tissue Differentiation; Tissues; Trans-Activators; Transgenes; Transgenic Mice; Work; adult stem cell; c-myc Genes; cancer stem cell; cell motility; cell type; falls; genomic profiles; human disease; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insight; mouse development; mutant; new therapeutic target; novel; progenitor; programs; promoter; recruit; self-renewal; stem; stem cells; tissue regeneration; tissue stem cells; transcription factor; tumor

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.

项目摘要 P27是限制正常细胞增殖的CDK抑制剂。我们在T157显示了P27 C末端磷酸化 AKT的T198在中期G1和P27PT157PT198（P27PTPT）中增加了与新蛋白的相互作用 合作伙伴。我们最近显示C末端P27PTPT磷酸化促进其与CJUN的关联。 基因组分析显示p27与CJUN共同授予超过一半的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。靶基因抑制的曲线 由p27/cjun提出，p27ptpt反对组织分化。 P27/CJUN靶基因的子集与A结合 STAT3共识基序和P27，CJUN和STAT3似乎粘合并共同调节CMYC。尤其， P27PTPT是干细胞效力的驱动力：细胞P27PTPT上调SOX2，NANOG和CMYC球 和肿瘤在体内启动细胞。我们的TG-P27CK-DD小鼠是P27磷酸化突变体的转基因， 未能结合细胞周期蛋白/CDK，显示多器官过度生长并增加了大小，这表明P27CK-DD 废除WTP27的动作以限制正常的茎和祖细胞扩展。该赠款调查了 p27作为正常细胞和发育中的转录调节剂。我们的数据支持以下假设 WTP27在分化过程中扮演着重要的转录作用，以限制茎和祖细胞的扩展 各种组织。相反，在C末端磷酸化时，P27与CJUN，STAT3和其他因素相互作用 扩展或维持组织茎或祖细胞并反对分化。我们将比较P27WT， AIM1中的P27CDK和P27CK-DD MEF评估P27与CJUN和STAT3在染色质上的相互作用 控制整个细胞周期的基因表达，从G0到Mid G1和G1/S相变。我们将 研究增加C末端P27磷酸化是否会改变共同调节剂并选择靶基因选择以及 废除了静态细胞中p27的共抑制功能。在AIM2中，我们将主动转录 来自转录非活性异染色质的全染色质，并将Chip-Seq和Chip-Mass规格执行 识别染色质相关的p27相互作用组，涉及反式激活器与阻遏物复合物。 AIM3 将研究TG-P27CK-中的小鼠发育，组织分化以及成人茎和祖细胞 DD和TG-P27CK-小鼠。我们将测试P27CK-DD是否赋予MEFS和 破坏IPSC为胚胎体的分化。这项工作将阐明p27的新功能 磷酸化依赖性转录调节剂对组织干细胞的影响和电势有影响 再生医学中的应用。 p27的受控调制或p27靶基因在本文中识别的p27靶基因 可能提供扩展或再生血液学和其他组织的潜力。了解p27/cjun的方式 转录程序调节组织开发可能会确定要利用的新治疗靶标 在分化和生长控制的界面处，组织再生并照亮其他人类疾病。