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.

概括功能性胰岛素分泌β细胞的减少是所有形式的糖尿病的进展，强调解密分子途径的翻译相关性调节了调节的形成，生长和功能 β细胞。转录网络对于β细胞的正确发育，分化和扩展至关重要通过形成3D枢纽的胰岛增强剂，超级增强剂和主动启动子进行工作。同源域转录因子PDX1是开发过程中β细胞转录网络的关键成员产后β细胞功能。 PDX1以人类糖尿病的单基因形式突变，并在早期起着关键作用胰腺规范，器官大小的调节以及β细胞形成，增殖和身份。我们的初步数据表明，开发β细胞暴露于下核定位改变和PDX1的水平降低蛋白质在整个细胞周期中前进时。此外，升高的PDX1水平预防细胞周期进展，表明表达动态调节是有效的β细胞扩张所必需的。我们识别PDX1 C末端（AA 207-223）中本质上无序的蛋白质区域（IDPR）。 IDPR，通常在转录因素中发现，缺乏固定的二级结构，并且可以适合柔性构象和相分离。 IDPR促进了超级蛋白质 - 蛋白质相互作用和转录中心的形成协调基因调控所需的增强子。我们发现PDX1 C末端培养基与多核胰腺祖细胞中的一个切割同源域转录因子OC1相互作用建立内分泌基因程序，对产后功能和β细胞补偿有长期影响。我们以前以前将E3泛素连接酶底物适配器SPOP鉴定为PDX1 C末端合作伙伴（通过 AA224-238）介导PDX1的泛素化和蛋白酶体降解。我们的初步数据暗示该SPOP和OC1竞争与PDX1 C末端的相互作用，并且OC1保护PDX1免受Spop- 介导的降解。值得注意的是，C末端具有几个糖尿病相关的人类突变，其中之一我们最近发现这破坏了PDX1/SPOP相互作用。那就是我们假设PDX1/OC1 相互作用部分由IDPR介导的部分调节PDX1稳定性，细胞周期进程和胰腺内分泌区分。该假设将以3个目的进行检验：（1）确定机制 PDX1和OC1合作以建立允许内分泌区分的染色质景观和增殖; （2）定义PDX1和OC1 IDPR在蛋白质 - 蛋白质相互作用和胰腺中的作用发展; （3）定义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的蛋白质稳定性和功能。人类的影响在这种情况下，将研究与糖尿病相关的突变。我们的研究将决定一部小说， PDX1 C末端在β细胞发育中未研究的结构特征的内聚作用。我们的结果研究将为优化基于细胞的疗法的β细胞扩展的治疗努力提供信息。