Defining the Role of MafA in Islet Beta Cells

描述（由申请人提供）：我们正在努力了解细胞形成和功能背后的分子程序，以便为新的糖尿病治疗策略提供治疗见解。这些研究表明，在2型糖尿病小鼠模型中，MafA转录因子既是成熟细胞功能的重要调节因子，又是失活的。MafA和在胰岛细胞中表达的唯一另一个密切相关的Maf蛋白MafB，最初是在我们的实验室和其他地方发现的，因为它们各自在刺激胰岛（即MafA，胰岛素）和细胞（MafB，胰高血糖素）激素基因转录方面具有重要意义。值得注意的是，MafA合成已被证明对功能细胞的产生至关重要，在胰岛素+细胞中存在MafB与未成熟和相对功能失调的细胞有关。我们对MafA和MafB蛋白嵌合体的分析表明，c端亮氨酸拉链（二聚化）区域在细胞基础实验中调节了MafA的独特内源性胰岛素基因激活能力，以及体外磷酸化依赖性DNA结合。我们假设这些序列也提供了MafA在体内的独特激活特性。这一假设将通过分析表达MafB/ a嵌合体或MafB的转基因对MafA /fl小鼠胰岛细胞功能的功能特征来验证，由于转基因Cre重组酶在MafA /fl小鼠中的作用，整个胰腺缺乏MafA表达。引人注目的是，通过在细胞中表达MafA和MafB，发现人类胰岛与啮齿动物不同。由于在物种之间没有观察到胰岛富集转录因子分布的其他差异，我们对MafB转基因小鼠的分析也将深入了解该因子是否有助于观察到人类和啮齿动物胰岛之间葡萄糖感知和胰岛素分泌特征的变化。我们假设MafA c端序列为转录协调节蛋白创造了独特的结合表面。令人惊讶的是，关于这些蛋白质在胰岛细胞发育或功能中所起作用的信息很少。我们已经实施了一种“细胞内”化学交联策略，从小鼠细胞中分离和表征这些分子。令人兴奋的初步数据表明，MafA能够结合常染色质混合谱系白血病2 （MLL2）组蛋白3赖氨酸4三甲基化复合物的组分MLL2、Rbbp5、Ash2、Dpy30和Wdr5。实验旨在确定MLL2如何影响mafa介导的基因激活和胰岛细胞功能。这里产生的数据将为转录调控机制提供有价值的见解，这可能会对糖尿病治疗的细胞疗法的生产产生影响。

DESCRIPTION (provided by applicant): A concerted effort is being made to understand the molecular programs underlying ¿ cell formation and function in order to provide therapeutic insight into new diabetes treatment strategies. From these studies, the MafA transcription factor has been demonstrated to be both an essential regulator of mature ¿ cell function, and inactivated in mouse models of type 2 diabetes mellitus. MafA and the only other closely related large Maf protein expressed in islet cells, MafB, were originally characterized in our laboratory and elsewhere due to their respective significance in stimulating islet ¿ (i.e. MafA, insulin) and a cell (MafB, glucagon) hormone gene transcription. Significantly, MafA synthesis has been shown to be critical to the production of functional ¿ cells, with the presence of MafB in insulin+ cells associated with immature and relatively dysfunctional cells. Our analyses of MafA and MafB protein chimeras have shown that the C-terminal leucine- zipper (dimerization) region regulates the distinct endogenous insulin gene activation ability of MafA in cell-based assays, as well as in phosphorylation-dependent DNA binding in vitro. We hypothesize that these sequences also provide distinguishing activating properties to MafA in vivo. This hypothesis will be tested by analyzing the functional characteristics of transgenes expressing either a MafB/A chimera or MafB on islet ¿ cell function in MafA(panc mice, which lack MafA expression throughout the pancreas due to the actions of a transgenic Cre recombinase in MafAfl/fl mice. Strikingly, human islets were found to be unlike rodents by expressing MafA and MafB in ¿ cells. Since no other differences were observed in islet-enriched transcription factor distribution between species, our analysis of MafB transgenic mice will also provide insight into whether this factor contributes to observed changes in glucose sensing and insulin secretion characteristics between human and rodent islets. We hypothesize that the MafA C-terminal sequences create unique binding surfaces for transcriptional coregulatory proteins. Surprisingly, there is a paucity of information regarding the role these proteins play in islet ¿ cell development or function. We have implemented an 'in cell' chemical cross-linking strategy to isolate and characterize such molecules from mouse ¿ cells. Exciting preliminary data illustrate the ability of MafA to bind MLL2, Rbbp5, Ash2, Dpy30, and Wdr5, components of the euchromatin mixed lineage leukemia 2 (MLL2) histone 3 lysine 4 trimethylation complex. Experiments are proposed to determine how MLL2 influences MafA-mediated gene activation and islet ¿ cell function. The data generated here will provide valuable insight into the transcriptional regulatory mechanisms that will likely be impactful in the production of cellular therapeutics for diabetes treatment. PUBLIC HEALTH RELEVANCE: A compelling body of evidence indicates that the MafA transcription factor represents a barometer of islet ¿ cell functionality. Our proposed studies will focus on determining how MafA together with its transcriptional coregulators impact islet ¿ cell biology. We believe that our findings will be essential to the ongoing efforts to generate ¿ cells from ES, iPS and/or adult cell sources for T1DM treatment.