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Functional interaction of transcriptional regulators in endocrine lineage specification

内分泌谱系规范中转录调节因子的功能相互作用

基本信息

批准号：
10577702
负责人：
Maureen A Gannon
金额：
$ 75.47万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10577702
关键词：
3-Dimensional ATAC-seq Beta Cell Biophysics C-terminal Cell Cycle Cell Cycle Progression Cell Line Cell Therapy Cell physiology Chromatin Compensation Complex Consensus Data Development Diabetes Mellitus Disease Endocrine Enhancers Epigenetic Process Exhibits Failure Gene Expression Regulation Genes Genetic Genetic Transcription Glucose Growth Heterozygote Human In Vitro Intervention Islets of Langerhans Mediating Microscopy Molecular Molecular Conformation Mus Mutagenesis Mutate Mutation Non-Insulin-Dependent Diabetes Mellitus Organ Size Organogenesis Pancreas Pathway interactions Phase Phenotype Phosphorylation Site Play Post-Translational Protein Processing Proliferating Property Protein Region Proteins Publishing Regulation Resolution Role Site Specific qualifier value Stimulus Structure Structure of beta Cell of islet Techniques Testing Therapeutic Ubiquitination Work design endocrine pancreas development flexibility genome editing homeodomain human embryonic stem cell human pluripotent stem cell in vivo insulin promoter factor 1 insulin secretion intermolecular interaction islet islet stem cells member mimetics mouse model multiple omics novel overexpression pancreas development permissiveness postnatal prediction algorithm prevent progenitor programs promoter protein protein interaction response single nucleus RNA-sequencing single-cell RNA sequencing stem cell differentiation transcription factor ubiquitin-protein ligase

项目摘要

SUMMARY Reduction in functional insulin-secreting β cells underlies the progression of all forms of diabetes, underscoring the translational relevance of deciphering molecular pathways regulating the formation, growth, and function of β cells. The transcriptional networks critical for the proper development, differentiation, and expansion of β cells work through islet enhancers, super enhancers, and active promoters that form 3D hubs. The homeodomain transcription factor Pdx1 is a critical member of the β-cell transcriptional network during development and in postnatal β-cell function. Pdx1 is mutated in monogenic forms of human diabetes and plays critical roles in early pancreas specification, regulation of organ size, and in β-cell formation, proliferation, and identity. Our preliminary data reveal that developing β cells exhibit altered subnuclear localization and reduced levels of Pdx1 protein as they advance through the cell cycle. Further, ectopically elevated levels of Pdx1 prevent cell cycle progression, suggesting that dynamic regulation of expression is required for effective β-cell expansion. We identify an intrinsically disordered protein region (IDPR) in the Pdx1 C-terminus (aa 207-223). IDPRs, commonly found within transcription factors, lack fixed secondary structure and are amenable to flexible conformations and phase separation. IDPRs promote protein-protein interactions and transcriptional hub formation at super enhancers necessary for coordinated gene regulation. We have found that the Pdx1 C-terminus mediates interaction with the one cut homeodomain transcription factor Oc1 in multipotent pancreatic progenitor cells to establish the endocrine gene program, with long-term impact on postnatal islet function and β-cell compensation. We previously identified the E3 ubiquitin ligase substrate adaptor SPOP as a PDX1 C-terminus partner (via aa224-238) that mediates ubiquitination and proteasomal degradation of PDX1. Our preliminary data suggest that SPOP and Oc1 compete for interaction with the Pdx1 C-terminus and that Oc1 protects Pdx1 from SPOP- mediated degradation. Notably, the C-terminus harbors several diabetes-associated human mutations, one of which we recently found disrupts the PDX1/SPOP interaction. Thus, we hypothesize that PDX1/OC1 interactions, in part mediated by their IDPRs, regulate Pdx1 stability, cell cycle progression, and pancreatic endocrine differentiation. This hypothesis will be tested in 3 Aims: (1) To determine the mechanisms whereby Pdx1 and Oc1 cooperate to establish a chromatin landscape permissive for endocrine differentiation and proliferation; (2) To define the roles of the Pdx1 and Oc1 IDPRs in protein-protein interaction and pancreas development; and (3) To define the molecular mechanisms by which the Pdx1 C-terminal domain regulates protein stability and function during pancreas organogenesis and endocrine differentiation. The impact of human diabetes-associated mutations will be investigated in this context. Our studies will determine a novel and cohesive role for unstudied structural features of the Pdx1 C-terminus in β-cell development. Results of our studies will inform therapeutic efforts to optimize β-cell expansion for cell-based therapies.

摘要功能性胰岛素分泌β细胞的减少是所有形式糖尿病进展的基础，强调破译调控细胞形成、生长和功能的分子途径的翻译相关性 β细胞。对β细胞的正常发育、分化和扩增至关重要的转录网络通过形成3D枢纽的胰岛增强剂、超级增强剂和活跃的促进剂来工作。同源域转录因子Pdx1是β细胞转录网络中的一个重要成员，在发育和发育中起重要作用。出生后β细胞功能。PDX1在人类糖尿病的单基因形式中发生突变，并在早期发挥关键作用胰腺的规范，器官大小的调节，以及在β中-细胞的形成，增殖和鉴定。我们的初步数据显示，发育中的β细胞表现出亚核定位改变和Pdx1水平降低蛋白质在细胞周期中前进。此外，异位升高的Pdx1水平阻止了细胞周期进展，这表明表达的动态调节是有效的β细胞扩增所必需的。我们鉴定Pdx1 C末端(AA 207-223)的一个固有无序蛋白区域(IDPR)。IDPR，通常发现于转录因子中，缺乏固定的二级结构，易于灵活构象和相分离。IDPR促进蛋白质-蛋白质相互作用和转录中心的形成协调基因调控所必需的增强剂。我们发现Pdx1的C-末端在在多能胰腺祖细胞中与单切同源结构域转录因子OC1的相互作用建立内分泌基因计划，对出生后胰岛功能和β细胞代偿产生长期影响。我们先前鉴定了E3泛素连接酶底物接头SPOP为PDX1的C末端伙伴(VIA Aa224-238)，介导PDX1的泛素化和蛋白酶体降解。我们的初步数据显示 SPOP和Oc1竞争与Pdx1 C末端的相互作用，并且Oc1保护Pdx1免受SPOP- 中介降级。值得注意的是，C末端含有几个与糖尿病相关的人类突变，其中之一我们最近发现，这会中断PDX1/SPOP的相互作用。因此，我们假设PDX1/OC1 相互作用部分由其IDPR介导，调节Pdx1的稳定性、细胞周期进展和胰腺内分泌分化。这一假设将在三个目标中得到检验：(1)确定 PDX1和OC1合作建立允许内分泌分化的染色质景观增殖；(2)确定Pdx1和OC1IDPR在蛋白质相互作用和胰腺中的作用发展；以及(3)确定Pdx1 C-末端结构域调节的分子机制胰腺器官发生和内分泌分化过程中蛋白质的稳定性和功能。人类的影响糖尿病相关突变将在此背景下进行调查。我们的研究将决定一部小说和未研究的Pdx1C末端结构特征在β细胞发育中的凝聚作用。我们的结果是研究将为优化基于细胞治疗的β细胞扩增的治疗努力提供信息。