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.

总结 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， 翻译相关的破译分子途径调节的形成，生长和功能， β细胞。转录网络对β细胞的正常发育、分化和扩增至关重要 通过胰岛增强子、超级增强子和形成3D枢纽的活性启动子发挥作用。同源结构域 转录因子Pdx 1是发育过程中β细胞转录网络的关键成员， 出生后的β细胞功能。PDX 1在人类糖尿病的单基因形式中突变，并在糖尿病的早期发病中起关键作用。 胰腺特化、器官大小调节以及β细胞形成、增殖和身份。我们 初步数据显示，发育中的β细胞表现出改变的亚核定位和降低的Pdx 1水平 蛋白质，因为他们通过细胞周期前进。此外，异位升高的Pdx 1水平阻止细胞周期 这表明表达的动态调节是有效的β细胞扩增所需的。我们 鉴定Pdx 1 C-末端（aa 207-223）的固有无序蛋白区（IDPR）。IDPR通常 在转录因子中发现，缺乏固定的二级结构，并适合于灵活的构象， 相分离IDPR促进蛋白质-蛋白质相互作用和转录枢纽的形成， 协调基因调控所必需的增强子。我们已经发现Pdx 1 C-末端介导了 在多能胰腺祖细胞中与单切割同源结构域转录因子Oc 1相互作用， 建立内分泌基因程序，对出生后胰岛功能和β细胞代偿有长期影响。 我们先前鉴定了E3泛素连接酶底物衔接子SPOP作为PDX 1 C-末端配偶体（通过 aa 224 -238），其介导PDX 1的泛素化和蛋白酶体降解。我们的初步数据显示 SPOP和Oc 1竞争与Pdx 1 C-末端的相互作用，Oc 1保护Pdx 1免受SPOP的影响。 介导的降解。值得注意的是，C-末端含有几种糖尿病相关的人类突变，其中一种是 我们最近发现它破坏了PDX 1/SPOP的相互作用。因此，我们假设PDX 1/OC 1 部分由IDPR介导的相互作用调节Pdx 1稳定性、细胞周期进程和胰腺癌。 内分泌分化这一假设将在3个目标进行测试：（1）确定机制， Pdx 1和Oc 1合作建立允许内分泌分化的染色质景观， （2）明确Pdx 1和Oc 1 IDPR在蛋白质-蛋白质相互作用和胰腺细胞增殖中的作用 （3）明确Pdx 1 C端结构域调控Pdx基因表达的分子机制 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研究。我们的研究将决定一部小说， Pdx 1 C-末端在β细胞发育中未研究的结构特征的内聚作用。成效 这些研究将为优化用于基于细胞的疗法的β细胞扩增的治疗努力提供信息。