We request R21 funding for a high risk, exploratory project for the genome-wide identification of target genes of PTF1a, a key bHLH transcriptional regulator of pancreatic organogenesis. During development, PTF1a functions in two temporally distinct trimeric complexes. It is required for early growth and morphogenesis in one complex bound to RBPJ, in a novel Notch-independent role. At the onset of islet, ductal and acinar lineage specification, it becomes restricted to nascent acinar cells and exchanges RBPJ for RBPJL, a constitutively active, vertebrate-specific and Notch-indifferent paralogue, and directs the acinar differentiation program. Comprehensive identification of direct targets of transcription factors that control embryonic development has proven extraordinarily difficult, due largely to the difficulty of recognizing functional binding sites in chromatin. Consequently, only limited advances in this area have been made, mostly through candidate, target-by-target strategies. The availability of only very small quantities of embryonic chromatin requires special technical considerations and refinements of current protocols. Here we propose an aggressive approach using cutting edge technologies to make a more substantial advance in highly inclusive target-identification during embryonic development using PTF1a in the pancreas as our model.
Aim 1 will establish conditions to prepare sufficient ChIP-enriched embryonic chromatin for massive parallel sequencing using the Illumina platform, identify and validate PTF1a-target sites, and compare targets for the early growth, lineage-decision, and differentiation stages of pancreatic development.
Aim 2 will compare and contrast the targets of the trimeric PTF1 complexes with those for RBPJL alone and for RBPJ in its distinct function for Notch-signaling. For reasons of high risk and the potential for high impact, we believe that the project outlined in this application is an appropriate candidate for the R21 mechanism.
Diabetes, pancreatitis and cancer are common diseases of the pancreas. By better understanding the control of normal development of the pancreas, we hope to contribute to therapies that may help reconstitute insulin producing cells, regenerate a pancreas badly damaged by pancreatitis, or identify ways to stop the growth of cancer cells derived from the exocrine pancreas without damaging other cells of the body.
| Meredith, David M; Borromeo, Mark D; Deering, Tye G et al. (2013) Program specificity for Ptf1a in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility. Mol Cell Biol 33:3166-79 |
| Masui, Toshihiko; Swift, Galvin H; Deering, Tye et al. (2010) Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells. Gastroenterology 139:270-80 |
