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.
Sox9 is a developmentally important transcription factor that controls the fate of pancreatic progenitor cells. I seek to identify new Sox9 target genes in order to better understand the regulation of these progenitors as they differentiate to endocrine cells, with the ultimate goal of improving treatments for type 1 diabetes.
|Huang, Wei; Wang, Guangliang; Delaspre, Fabien et al. (2014) Retinoic acid plays an evolutionarily conserved and biphasic role in pancreas development. Dev Biol 394:83-93|