Transcriptional Coregulators in Pancreas Development

描述（由申请人提供）：分泌胰岛素的胰岛细胞的失败是1型和2型糖尿病的标志，导致本实验室高度关注调节胰岛细胞发育和功能的分子机制。我们先前在酵母双杂交筛选中鉴定了Pcif 1，以寻找Pdx 1的伴侣，Pdx 1是一种同源结构域转录因子和人糖尿病基因，在转录层次中枢转地定位，该转录层次控制着细胞团的发育、成年细胞的功能和存活以及成年细胞的能力， 补偿由胰岛素抵抗强加的增加的代谢需求。在之前的资助期间，我们确定Pcif 1至少部分地通过其作为底物适配器的作用将Pdx 1招募到基于cullin 3的复合物中进行泛素化和蛋白酶体降解，从而暗示Pcif 1是调节Pdx 1水平的靶点。事实上，Pcif 1在细胞中的功能丧失会升高Pdx 1蛋白，并刺激重要的Pdx 1转录靶点的表达，包括胰岛素、MafA和Glut 2。Pcif 1的体内遗传破坏揭示了在胚胎发育过程中通过Pdx 1依赖性和可能的Pdx 1独立性机制调节成体细胞更新和功能以及内分泌谱系分配的作用。我们假设Pcif 1通过其调节关键转录调节因子的水平/功能的能力来关键地调节胚胎细胞形成和成体细胞周期。这一假设将在两个目标中得到检验：目标1。为了确定Pcif 1如何调节成体细胞的周转，目的2。确定Pcif 1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靶向治疗提供概念性支架，这些靶向治疗干扰Pcif 1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，并可能应用于涉及干细胞分化为细胞、细胞重编程和细胞凋亡的方法。来自其他成熟细胞类型的细胞，以及促进糖尿病患者内源性β细胞群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的失败是1型和2型糖尿病的标志，促使人们努力开发用于替代疗法的胰岛素产生细胞的新来源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接头Pcif 1在细胞生物学中的功能的重要问题。获得的见解将为开发靶向治疗提供概念性支架，这些治疗干扰Pcif 1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，并可能应用于干细胞分化，细胞重编程和促进糖尿病患者内源性细胞质量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.