Ldb1-mediated transcriptional complexes during beta-cell development and function

β 细胞发育和功能过程中 Ldb1 介导的转录复合物

基本信息

批准号：
9110565
负责人：
Chad S Hunter
金额：
$ 7.35万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9110565
关键词：
Adult Affect American Automobile Driving Beta Cell Binding Binding Proteins Biological Assay Cell Count Cell Line Cell Therapy Cell physiology Cells Complex DNA Binding Data Development Developmental Process Diabetes Mellitus Disease Ductal Epithelial Cell Economic Burden Electrophoretic Mobility Shift Assay Embryo Endocrine Excision Exhibits Family Functional disorder Funding Future Gene Expression Gene Targeting Generations Genetic Programming Genetic Transcription Hormones Immunofluorescence Immunologic Immunoprecipitation In Situ In Vitro Insulin Intervention Islet Cell Islets of Langerhans Knockout Mice Knowledge LIM Domain Ligation Link Mass Spectrum Analysis Mediating Modeling Mus Mutant Strains Mice Organogenesis Outcome Pancreas Patients Pattern Phenocopy Phenotype Population Production Proteins Publishing Quality of life Reporter Reporter Genes Role SS DNA BP Small Interfering RNA Specific qualifier value Stem cells Structure of beta Cell of islet Testing Tissues Transactivation Work base blood glucose regulation cell type combat comparative conditional mutant developmental genetics diabetes mellitus therapy diabetic endocrine pancreas development experience health economics homeodomain improved in vivo interest islet knock-down neural patterning pancreas development postnatal progenitor programs protein expression public health relevance research study success transcription factor

项目摘要

DESCRIPTION (provided by applicant): Pancreatic islets of Langerhans contain insulin-secreting beta cells required for maintaining glucose homeostasis. Dysfunction in beta cell activity or survival results in diabetes mellitus, a disease currently affecting millions of Americans with numbers expected to greatly increase. This is creating an enormous economic and health burden. A future strategy for improving diabetic outcomes will include cell-based therapies wherein functional beta cells are generated to replenish those lost during diabetes progression. Success will require better understanding the complex transcriptional programs that specify and differentiate functional beta cells from progenitors. We previously showed that the LIM domain transcription factor, Islet-1, is required for islet endocrine cell development and function. Furthermore, the activity of Islet-1 during late pancreas development requires the interacting coregulator, Ldb1. My published study defined the pancreatic Ldb1 expression pattern and roles in developing endocrine cells. Whereas mice lacking Ldb1 in the developing islets exhibited a phenotype largely overlapping with Islet-1, it appears that Ldb1 also acts independently in early progenitor cells. Furthermore, interacting factors that comprise the beta cell Ldb1-Islet-1 complex remain unknown, although preliminary data suggests that the complexes are very large. In other tissues, Ldb1 binds the SSBP class of coregulators (or Single Stranded DNA-Binding Proteins, including SSBP2-4). These factors impact the transactivation capacity and stability of Ldb1 complexes. However, nothing is known of SSBP expression, interaction or function in islet beta cells. Data generated from my K01 studies utilized immunoprecipitation and mass spectrometry to isolate and identify beta cell line Ldb1 and Islet-1 binding proteins. The SSBP3 proteins appeared on the candidate list, suggesting that they participate in pancreas Ldb1 complexes. My R03 Aims will define roles for Ldb1 complexes during early pancreas progenitor cells as well as in adult beta cells. Aim 1 will utilize a conditional Ldb1 mutant mouse (Pdx1-Cre;Ldb1F/-) to test for Ldb1 impacts during early pancreas progenitors. These cells are largely devoid of Islet-1, suggesting roles for another Ldb1- interacting LIM transcription factor. The early Ldb1 knockout mice are expected to (at least) phenocopy our published endocrine-specific Ldb1 model, plus have additional impacts on progenitor formation, proliferation, or cell-type allocation. Aim 2 examines the function of Ldb1-interacting SSBP3 coregulators in beta cells. Approaches will include ChIP, siRNA knockdown, immunofluorescence, reporter gene assays and DNA- binding experiments to examine how SSBP factors impact Ldb1-Islet-1 complexes. With this R03 proposal, I will further test my central hypothesis that Ldb1 complexes are required for pancreatic organogenesis and beta cell function. My extensive experience with LIM factor complexes make me uniquely suited to executing these Aims, which have the promise of generating interesting preliminary data toward R01 funding.

描述（由申请人提供）：胰岛含有维持葡萄糖稳态所需的胰岛素分泌β细胞，β细胞活性或存活功能障碍会导致糖尿病，这种疾病目前影响着数百万美国人，预计其数量将大大增加。正在造成巨大的经济和健康负担，改善糖尿病结果的未来策略将包括基于细胞的疗法，尽管功能性β细胞的产生是为了补充糖尿病进展过程中损失的细胞，但成功需要更好地了解这一复杂的过程。我们之前表明，LIM 结构域转录因子 Islet-1 是胰岛内分泌细胞发育和功能所必需的。此外，胰岛发育后期的 Islet-1 活性也需要。我发表的研究定义了胰腺 Ldb1 的表达模式和在发育中的内分泌细胞中的作用，而发育中的胰岛中缺乏 Ldb1 的小鼠表现出与发育中的胰岛细胞很大程度上重叠的表型。 Islet-1，即 Ldb1 也在早期祖细胞中独立发挥作用。此外，组成 β 细胞 Ldb1-Islet-1 复合物的相互作用因子仍然未知，尽管初步数据表明 Ldb1 在其他组织中结合得非常大。 SSBP 类共调节剂（或单链 DNA 结合蛋白，包括 SSBP2-4）。然而，这些因素影响 Ldb1 复合物的反式激活能力和稳定性。我对胰岛 β 细胞中的 SSBP 表达、相互作用或功能一无所知。我的 K01 研究利用免疫沉淀和质谱法分离和鉴定了 β 细胞系 Ldb1 和 Islet-1 结合蛋白。 SSBP3 蛋白出现在候选列表中。表明它们参与胰腺 Ldb1 复合物，My R03 Aims 将定义 Ldb1 复合物在早期胰腺祖细胞以及成人 β 细胞中的作用。将利用条件性 Ldb1 突变小鼠 (Pdx1-Cre;Ldb1F/-) 用于测试 Ldb1 对早期胰腺祖细胞的影响。这些细胞基本上缺乏 Islet-1，表明另一种与 Ldb1 相互作用的 LIM 转录因子在早期 Ldb1 敲除小鼠中发挥作用。预计（至少）对我们发表的内分泌特异性 Ldb1 模型进行表型复制，并且对祖细胞形成、增殖、目标 2 检查 β 细胞中 Ldb1 相互作用的 SSBP3 共调节子的功能，方法包括 ChIP、siRNA 敲低、免疫荧光、报告基因检测和 DNA 结合实验，以检查 SSBP 因子如何影响 Ldb1-Islet-。通过这个 R03 提案，我将进一步检验我的中心假设，即 Ldb1 复合物是胰腺器官发生和 β 细胞功能所必需的。 LIM 因子复合体使我特别适合执行这些目标，这些目标有望为 R01 资金生成有趣的初步数据。