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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 p27 是一种限制正常细胞增殖的 CDK 抑制剂。我们在 T157 处显示了 p27 C 末端磷酸化 AKT 在 G1 中期增加 T198，p27pT157pT198 (p27pTpT) 促进与新蛋白质的相互作用合作伙伴。我们最近表明 C 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。被抑制的靶基因概况 p27/cJun 表明 p27pTpT 反对组织分化。 p27/cJun 靶基因的子集与 STAT3 共有基序以及 p27、cJun 和 STAT3 似乎都结合并共同调节 cMYC。尤其， p27pTpT 是干细胞效力的驱动因素：细胞 p27pTpT 上调球体、SOX2、NANOG 和 cMYC 和体内肿瘤起始细胞。我们的 TG-p27CK-DD 转基因 p27 拟磷突变体小鼠无法结合细胞周期蛋白/CDK，显示多器官过度生长和大小增加，表明 p27CK-DD 废除 WTp27 抑制正常茎和祖细胞扩张的作用。这笔赠款调查了 p27 作为正常细胞和发育中的转录调节因子。我们的数据支持以下假设： WTp27 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 端磷酸化后，p27 与 cJun、STAT3 和其他因子相互作用扩大或维持组织干细胞或祖细胞并反对分化。我们将比较 p27WT， AIM1 中的 p27CDK 和 p27CK-DD MEFS 评估 p27 如何与染色质上的 cJun 和 STAT3 相互作用控制从 G0 到 G1 中期以及 G1/S 相变的整个细胞周期的基因表达。我们将研究 C 末端 p27 磷酸化的增加是否会改变辅助调节因子和靶基因的选择以及消除静止细胞中 p27 的共抑制功能。在AIM2中，我们将主动转录的分开从转录失活的异染色质中分离出常染色质，并进行 ChIP-seq 和 ChIP-Mass Spec 鉴定参与反式激活蛋白与阻遏蛋白复合物的染色质相关 p27 相互作用组。目标3 将研究 TG-p27CK- 中的小鼠发育、组织分化以及成体干细胞和祖细胞群 DD 和 TG-p27CK- 小鼠。我们将测试 p27CK-DD 是否赋予 MEF 更强的重编程潜力破坏 iPSC 向拟胚体的分化。这项工作将阐明 p27 作为磷酸化依赖性转录调节因子对组织干细胞控制和潜力的影响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，可能具有扩大或再生血液和其他组织的潜力。了解 p27/cJun 如何转录程序调节组织发育可能会识别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。