Transcriptional coregulators in pancreas development

描述(由申请人提供)：胰岛素分泌胰岛细胞的失败是1型和2型糖尿病的标志，导致本实验室将重点放在调节胰腺细胞发育和功能的分子机制上。我们之前在酵母双杂交筛选中发现了Pcif1，以寻找Pdx1的合作伙伴，Pdx1是一种同源结构域转录因子和人类糖尿病基因，位于调控细胞团的发育、成年细胞的功能和生存以及成年细胞补偿胰岛素抵抗所带来的代谢需求增加的能力的转录层次中。在之前的资助期间，我们确定Pcif1至少部分地通过其底物适配器的角色将Pdx1招募到基于cullin3的复合体中进行泛素化和蛋白酶体降解，从而使Pcif1成为调节Pdx1水平的靶标。事实上，Pcif1在细胞中的功能丧失会上调Pdx1蛋白，并刺激重要的Pdx1转录靶标的表达，包括胰岛素、Mafa和Glut2。在体内，Pcif1的遗传干扰揭示了在胚胎发育过程中，通过依赖于Pdx1和可能不依赖于Pdx1的机制，Pcif1在调节成体细胞更新和功能以及内分泌谱系分配中的作用。我们假设，Pcif1通过调节关键转录调节因子的水平/功能来关键地调节胚胎细胞的形成和成年细胞的周期。这一假说将在两个方面得到验证：目的1.确定Pcif1如何调节成年细胞的周转；2.确定Pcif1如何影响胚胎胰腺发育过程中细胞谱系的出现。从拟议的实验中获得的见解将为开发干扰Pcif1的表达或其与细胞中关键底物的相互作用以影响细胞质量和细胞功能的靶向治疗提供概念支架，潜在地应用于涉及干细胞向细胞分化的方法、其他成熟细胞类型的细胞重新编程以及促进糖尿病患者内源性β细胞质量的功能和再生的方法。 公共卫生相关性：分泌胰岛素的胰岛细胞失效是1型和2型糖尿病的标志，促使人们加紧努力开发新的胰岛素产生细胞来源，用于替代疗法和促进内源性细胞再生的新方法。拟议的研究将解决有关泛素连接酶底物适配器Pcif1在细胞生物学中的功能的重要问题。所获得的见解将为靶向疗法的开发提供概念框架，该疗法可以干扰Pcif1的表达或其与细胞中关键底物的相互作用，从而影响细胞群和细胞功能，并有可能应用于干细胞分化、细胞重新编程以及促进糖尿病患者内源性细胞群的功能和再生的方法。

DESCRIPTION (provided by applicant): Failure of insulin secreting pancreatic islet ¿ cells is a hallmark of both type1 and type 2 diabetes, leading to this laboratory's intense focus on molecular mechanisms regulating pancreatic ¿ cell development and function. We previously identified Pcif1 in a yeast two hybrid screen for partners of Pdx1, a homeodomain transcription factor and human diabetes gene pivotally positioned in the transcriptional hierarchy governing the development of ¿ cell mass, the function and survival of adult ¿ cells, and the ability of adult ¿ cells to compensate for increased metabolic demand imposed by insulin resistance. During the previous funding period we determined that Pcif1 acts at least in part through its role as a substrate adaptor to recruit Pdx1 into a cullin3-based complex for ubiquitination and proteasomal degradation, thereby implicating Pcif1 as a target for regulating Pdx1 levels. Indeed, Pcif1 loss of function in ¿ cells elevates Pdx1 protein and stimulates expression of important Pdx1 transcriptional targets, including insulin, MafA and Glut2. In vivo genetic disruption of Pcif1 reveals roles in regulating adult ¿ cell turnover and function and in endocrine lineage allocation during embryonic development, through Pdx1-dependent and, likely, Pdx1-independent mechanisms. We hypothesize that Pcif1 critically regulates embryonic ¿ cell formation and the adult ¿ cell cycle through its ability to modulate the levels/functions of key transcriptional regulators. This hypothesis will be tested in two aims: Aim 1. To determine how Pcif1 regulates the turnover of adult ¿ cells, and Aim 2. To determine how Pcif1 influences the emergence of the ¿ cell lineage during embryonic pancreas development. Insights gained from the proposed experiments will provide a conceptual scaffold for the development of targeted therapeutics that interfere with Pcif1 expression or its interactions with critical substrates in the ¿ cell to influence ¿ cell mass and ¿ cell function, with potential application to approaches involving stem cell differentiation to ¿ cells, cellular reprogramming of ¿ cells from other mature cell types, and for approaches to promote the function and regeneration of endogenous beta cell mass in patients with diabetes. PUBLIC HEALTH RELEVANCE: Failure of insulin secreting pancreatic islet ¿ cells is a hallmark of both type1 and type 2 diabetes, prompting intense effort to develop new sources of insulin- producing ¿ cells for replacement therapies and new ways to promote endogenous ¿ cell regeneration. The proposed studies will address important questions about the functions of the ubiquitin ligase substrate adaptor Pcif1 in ¿ cell biology. Insights gained will provide a conceptual scaffold for the development of targeted therapeutics that interfere with Pcif1 expression or its interactions with critical substrates in the ¿ cell to influence ¿ cell mass and ¿ cell function, with potential application to stem cell differentiation, cellular reprogramming and approaches to promote the function and regeneration of endogenous ¿ cell mass in patients with diabetes.