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

 描述（由申请方提供）：胰岛含有维持葡萄糖稳态所需的胰岛素分泌β细胞。β细胞活性或存活的功能障碍导致糖尿病，这是一种目前影响数百万美国人的疾病，预计人数将大大增加。这造成了巨大的经济和健康负担。改善糖尿病预后的未来策略将包括基于细胞的治疗，其中产生功能性β细胞以补充糖尿病进展期间丢失的细胞。成功将需要更好地理解复杂的转录程序，这些程序指定并区分功能性β细胞和祖细胞。我们以前表明，LIM结构域转录因子，胰岛-1，是所需的胰岛内分泌细胞的发育和功能。此外，在胰腺发育后期，胰岛-1的活性需要相互作用的辅助调节因子Ldb 1。我发表的研究定义了胰腺Ldb 1表达模式和在发育中的内分泌细胞中的作用。尽管在发育中的胰岛中缺乏Ldb 1的小鼠表现出与Islet-1在很大程度上重叠的表型，但似乎Ldb 1在早期祖细胞中也独立发挥作用。此外，尽管初步数据表明复合物非常大，但构成β细胞Ldb 1-Islet-1复合物的相互作用因子仍然未知。在其他组织中，Ldb 1结合SSBP类辅调节因子（或单链DNA结合蛋白，包括SSBP 2 -4）。这些因素影响Ldb 1复合物的反式激活能力和稳定性。然而，对SSBP在胰岛β细胞中的表达、相互作用或功能一无所知。从我的K 01研究中产生的数据利用免疫沉淀和质谱分离和鉴定β细胞系Ldb 1和Islet-1结合蛋白。SSBP 3蛋白出现在候选列表中，表明它们参与胰腺Ldb 1复合物。我的R 03目标将定义Ldb 1复合物在早期胰腺祖细胞以及成人β细胞中的作用。目标1将利用 条件性Ldb 1突变小鼠（Pdx 1-Cre; Ldb 1F/-），以测试Ldb 1对早期胰腺祖细胞的影响。这些细胞在很大程度上缺乏Islet-1，这表明另一种Ldb 1相互作用的LIM转录因子的作用。早期Ldb 1基因敲除小鼠预计（至少）表型复制我们发表的内分泌特异性Ldb 1模型，加上对祖细胞形成，增殖或细胞类型分配的额外影响。目的2检测β细胞中Ldb 1相互作用的SSBP 3辅助调节因子的功能。方法将包括ChIP，siRNA敲低，免疫荧光，报告基因测定和DNA结合实验，以检查SSBP因子如何影响Ldb 1-胰岛-1复合物。通过这个R 03的提议，我将进一步验证我的中心假设，即Ldb 1复合物是胰腺器官发生和β细胞功能所必需的。我在LIM因子复合物方面的丰富经验使我特别适合执行这些目标，这些目标有望为R 01资金提供有趣的初步数据。

 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.