Determining the developmental progression of PTF1 targets by ChIP-seq

描述（由申请人提供）：我们申请R21资助一个高风险的探索性项目，用于PTF1a靶基因的全基因组鉴定，PTF1a是胰腺器官发生的关键bHLH转录调节因子。在发育过程中，PTF1a在两个时间上不同的三聚体复合体中起作用。它是一个与RBPJ结合的复合体的早期生长和形态发生所必需的，在一个新的notch独立的作用中。在胰岛、导管和腺泡谱系分化开始时，它被限制在新生的腺泡细胞中，并将RBPJ交换为RBPJL， RBPJL是一种构成活性的、脊椎动物特异性的、缺口无关的旁系，并指导腺泡分化程序。全面鉴定控制胚胎发育的转录因子的直接靶标已被证明是非常困难的，这主要是由于难以识别染色质中的功能结合位点。因此，在这方面只取得了有限的进展，主要是通过候选的逐个目标的战略。只有非常少量的胚胎染色质的可用性需要特殊的技术考虑和改进当前的方案。在这里，我们提出了一种积极的方法，使用尖端技术，以胰腺中的PTF1a为模型，在胚胎发育过程中高度包容性的靶标识别方面取得更实质性的进展。目的1将建立条件，利用Illumina平台制备足够的chip富集的胚胎染色质进行大规模平行测序，鉴定和验证ptf1a靶点，并比较胰腺发育的早期生长、谱系决定和分化阶段的靶点。Aim 2将比较和对比三聚体PTF1复合物与RBPJL单独的靶标以及RBPJ在notch信号传导中的独特功能。由于高风险和高影响的潜在原因，我们认为本申请中概述的项目是R21机制的合适候选者。公共卫生相关性：糖尿病、胰腺炎和癌症是胰腺的常见疾病。通过更好地了解胰腺正常发育的控制，我们希望有助于治疗，可能有助于重建胰岛素产生细胞，再生胰腺炎严重受损的胰腺，或找到方法来阻止来自外分泌胰腺的癌细胞的生长，而不损害身体的其他细胞。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.