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

• 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
• 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

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糖尿病进展的基础，强调 破译调节形成、生长和功能的分子途径的翻译相关性 β细胞。转录网络对于 β 细胞的正常发育、分化和扩增至关重要 通过形成 3D 中心的胰岛增强子、超级增强子和活性启动子发挥作用。同源域 转录因子 Pdx1 是 β 细胞转录网络在发育和发育过程中的重要成员。 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，并在早期糖尿病中发挥关键作用 胰腺规格、器官大小的调节以及 β 细胞的形成、增殖和身份。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 当它们在细胞周期中前进时蛋白质。此外，Pdx1 水平异位升高会阻止细胞周期 进展，表明表达的动态调节是β细胞有效扩增所必需的。我们 鉴定 Pdx1 C 末端 (aa 207-223) 中的本质无序蛋白质区域 (IDPR)。 IDPR，通常 在转录因子中发现，缺乏固定的二级结构，并且适合灵活的构象和 相分离。 IDPRs 促进蛋白质-蛋白质相互作用和超级转录中心的形成 协调基因调控所必需的增强子。我们发现 Pdx1 C 末端介导 与多能胰腺祖细胞中的单切同源域转录因子 Oc1 相互作用 建立内分泌基因程序，对出生后胰岛功能和β细胞代偿具有长期影响。 我们之前将 E3 泛素连接酶底物适配器 SPOP 确定为 PDX1 C 末端伙伴（通过 aa224-238) 介导 PDX1 的泛素化和蛋白酶体降解。我们的初步数据表明 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 末端含有多种与糖尿病相关的人类突变，其中之一 我们最近发现它破坏了 PDX1/SPOP 相互作用。因此，我们假设 PDX1/OC1 相互作用，部分由 IDPR 介导，调节 Pdx1 稳定性、细胞周期进程和胰腺 内分泌分化。这一假设将在 3 个目标中得到检验：(1) 确定其机制 Pdx1 和 Oc1 合作建立一个允许内分泌分化和 增殖; (2) 明确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) 明确 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 糖尿病相关突变将在这种背景下进行研究。我们的研究将确定一个新颖且 Pdx1 C 末端未经研究的结构特征在 β 细胞发育中的凝聚作用。我们的结果 研究将为优化基于细胞的疗法的β细胞扩增的治疗工作提供信息。