Investigation of Sox9 target genes in the pancreas

描述(申请人提供)：1型糖尿病(T1D)是由胰腺中产生胰岛素的细胞丢失引起的。供体胰岛体外扩增或诱导内源性胰祖细胞向内分泌方向分化可能是治疗T1D的有效方法。因此，了解胰腺祖细胞的调控是很重要的。我试图找出在治疗T1D时可以操纵的新途径。为了做到这一点，我提议在胰腺中识别新的Sox9靶基因。SOX9是一种转录因子，在包括胰腺祖细胞在内的几个干细胞和祖细胞系统中是分化的主要调节因子。在小鼠中，由于祖细胞增殖减少，Sox9零突变导致胰腺变小。在人类中，SOX9单倍体不足会导致一种称为钟状核显示的疾病，其特征包括胰岛形态改变和少数细胞。在斑马鱼中，sox9b在胰腺前体细胞中表达，是正常胰腺发育所必需的。Sox9b的表达受两条信号通路控制，这两条信号通路对胰腺祖细胞的调控也很重要。斑马鱼胰腺前体细胞受Notch信号调节，Notch信号控制这些细胞中sox9b的表达。我们最近发现，维甲酸(RA)信号是胰腺前体调节中的另一个重要途径，而在这些细胞中，sox9b是RA信号的靶点。因此，Sox9b可能在整合胰腺前体细胞中的Notch和RA信号方面发挥关键作用。有趣的是，RA处理人乳腺癌细胞会导致SOX9的上调。SOX9反过来直接调节HES1的表达，HES1是一个典型的Notch效应基因。我推测，Sox9b直接调节HES1的同源基因，这是HER基因家族的成员。在目标1中，我将采用候选方法来确定Sox9b在HER家族中的直接靶标。然后，我将使用突变株来研究这些靶基因在胰腺发育中的作用，其中许多突变株已经在我们的实验室获得。在一种平行的、不偏不倚的方法中，我将在人类胰腺癌细胞系PANC-1中识别SOX9靶点。首先，我将使用染色质免疫沉淀和高通量测序(CHIP-SEQ)相结合的方法来识别活性增强子，对照开放增强子的已知标记物EP300和公开可用的数据集。其次，我将使用生物信息学的方法来鉴定与这些增强子一致的可能的SOX9结合位点。第三，在PANC-1细胞和PANC-1细胞中，我将使用qRT-PCR检测可能的SOX9结合增强子两侧的基因表达。然后，我将使用荧光素酶报告实验测试SOX9对候选SOX9靶向增强子的调节。重要的是，上面概述的项目具有高度的协作性，将为我提供新领域的培训，如胰腺发育、基因组学和生物信息学。这项工作将辅之以数据分析和统计方面的课程。最终，我博士后培训的多学科性质将推动我进入我想要的职业生涯，成为一名学术PI。

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is caused by the loss of insulin-producing ?-cells in the pancreas. The in vitro expansion of donated islets for transplantation or the induction of endogenous pancreatic progenitors toward the endocrine lineage may be successful in the treatment of T1D. Therefore, an understanding of pancreatic progenitor regulation is important. I seek to identify new pathways that can be manipulated in the treatment of T1D. To do so I propose to identify novel Sox9 target genes in the pancreas. Sox9 is a transcription factor that is a master regulator of differentiation in several stem and progenitor cell systems, including pancreatic progenitors. In mice, null mutations in Sox9 lead to a small pancreas due to the decreased proliferation of progenitors. In humans, SOX9 haploinsufficiency leads to a condition called Campomelic displaysia, the features of which include altered islet morphology and few ? -cells. In zebra fish, sox9b is expressed in pancreatic progenitors and is required for normal pancreas development. sox9b expression is controlled by two signaling pathways also important for pancreas progenitor regulation. Zebra fish pancreas progenitors are regulated by Notch signaling, which controls sox9b expression in these cells. We have recently shown that retinoic acid (RA) signaling is another pathway important in pancreas progenitor regulation and that in these cells sox9b is a target of RA signaling. Thus, Sox9b may play a pivotal role in integrating both Notch and RA- signaling in pancreatic progenitors. Interestingly, RA treatment of human breast cancer cells leads to the up- regulation of SOX9. SOX9 in turn directly regulates the expression of HES1, a canonical Notch effector gene. I hypothesize that Sox9b directly regulates homologs of HES1, which are members of the her gene family. In Aim 1, I will take a candidate approach to identify direct targets of Sox9b within the her family. I will then investigate the role of these target genes in pancreas development using mutant lines, many of which are already available in our lab. In a parallel, unbiased approach, I will identify SOX9 targets in a human pancreatic carcinoma line, PANC-1 cells. First, I will identify active enhancers using a combination of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against a known marker of open enhancers, EP300, and publicly available datasets. Second, I will use a bioinformatics approach to identify putative SOX9 binding sites coincident with these enhancers. Third, in PANC-1 cells and PANC-1 cells transfected with shRNAs against SOX9, I will assay the expression of genes flanking putative SOX9-binding enhancers using qRT-PCR. I will then test the regulation of candidate SOX9-target enhancers by SOX9 using a luciferase reporter assay. Importantly, the project outlined above is highly collaborative and will provide me with training in fields new to me such as pancreas development, genomics and bioinformatics. The work will be supplemented with coursework in data analysis and statistics. Ultimately, the multidisciplinary nature of my postdoctoral training will propel me into my desired career as an academic PI.