Investigation of Sox9 target genes in the pancreas

胰腺中 Sox9 靶基因的研究

• 批准号: 8647249
• 负责人: Rebecca Beer
• 金额: $ 5.16万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647249
• 关键词: Antibodies; Binding; Binding Sites; Bioinformatics; Biological Assay; Breast Cancer Cell; Cancer cell line; Cell Differentiation process; Cell Line; Cells; Data; Data Analyses; Data Set; Development; Developmental Biology; Disease; EP300 gene; ES01; Endocrine; Enhancers; Family; Fishes; Gene Expression; Gene Expression Regulation; Gene Family; Gene Targeting; Genes; Genetic Enhancer Element; Genomics; Goals; Homologous Gene; Human; Human Cell Line; In Vitro; Institution; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans Transplantation; Knowledge; Lead; Luciferases; Methods; Morphology; Multipotent Stem Cells; Mus; Natural regeneration; Nature; Pancreas; Pancreatic carcinoma; Pathway interactions; Play; Regulation; Replacement Therapy; Reporter; Research; Role; Signal Pathway; Signal Transduction; Symptoms; System; Testing; Training; Transcriptional Regulation; Transgenic Organisms; Tretinoin; Undifferentiated; Up-Regulation; Work; Zebrafish; campomelic dysplasia; career; chromatin immunoprecipitation; design; improved; in vivo; inhibitor/antagonist; islet; member; multidisciplinary; mutant; notch protein; novel; null mutation; pancreas development; post-doctoral training; progenitor; public health relevance; research study; skills; statistics; stem; stem cells; success; tool; transcription factor

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.

描述（由申请人提供）：1型糖尿病（T1 D）是由胰岛素产生？胰腺中的细胞体外扩增供移植的胰岛或诱导内源性胰腺祖细胞向内分泌谱系分化可能成功治疗T1 D。因此，了解胰腺祖细胞的调节是很重要的。我试图确定新的途径，可以在T1 D的治疗操纵。为此，我建议在胰腺中鉴定新的Sox 9靶基因。Sox 9是一种转录因子，是几种干细胞和祖细胞系统（包括胰腺祖细胞）中分化的主要调节因子。在小鼠中，Sox 9的无效突变导致胰腺变小，这是由于祖细胞增殖减少。在人类中，SOX 9单倍不足导致一种称为Campomelic显示症的疾病，其特征包括胰岛形态改变和很少？- 细胞。在斑马鱼中，sox 9 b在胰腺祖细胞中表达，并且是正常胰腺发育所必需的。Sox 9 b的表达受两种信号传导途径控制，这两种信号传导途径对于胰腺祖细胞调节也是重要的。斑马鱼胰腺祖细胞受Notch信号转导调控，Notch信号转导控制这些细胞中的sox 9 b表达。我们最近发现，视黄酸（RA）信号是胰腺祖细胞调节的另一个重要途径，在这些细胞中，sox 9 b是RA信号的靶点。因此，Sox 9 b可能在胰腺祖细胞中整合Notch和RA信号传导中起关键作用。有趣的是，人乳腺癌细胞的RA治疗导致S 0X 9的上调。S 0X 9进而直接调节经典Notch效应基因HES 1的表达。我假设Sox 9 b直接调节HES 1的同源物，这是她的基因家族的成员。在目标1中，我将采用候选方法来确定Sox 9 b在其家族中的直接靶点。然后，我将使用突变株系研究这些靶基因在胰腺发育中的作用，其中许多已经在我们的实验室中获得。在一个平行的，无偏见的方法，我将确定SOX 9在人类胰腺癌细胞系，PANC-1细胞的目标。首先，我将使用染色质免疫沉淀结合高通量测序（ChIP-Seq）对开放增强子的已知标记EP 300和公开可用的数据集进行鉴定。其次，我将使用生物信息学的方法来确定与这些增强子一致的推定的SOX 9结合位点。第三，在PANC-1细胞和用针对SOX 9的shRNA转染的PANC-1细胞中，我将使用qRT-PCR测定推定的SOX 9结合增强子侧翼基因的表达。然后，我将使用荧光素酶报告基因测定来测试SOX 9对候选SOX 9靶增强子的调节。重要的是，上面概述的项目是高度合作的，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。这项工作将补充课程的数据分析和统计。最终，我的博士后培训的多学科性质将推动我进入我理想的职业生涯作为一个学术PI。