Abstract
The homeobox transcription factor Pdx-1 is necessary for pancreas organogenesis and beta cell function, however, most Pdx-1-regulated genes are unknown. To further the understanding of Pdx-1 in beta cell biology, we have characterized its genomic targets in NIT-1 cells, a mouse insulinoma cell line. To identify novel targets, we developed a microarray that includes traditional promoters as well as non-coding conserved elements, micro-RNAs, and elements identified through an unbiased approach termed serial analysis of chromatin occupancy. In total, 583 new Pdx-1 target genes were identified, many of which contribute to energy sensing and insulin release in pancreatic beta cells. By analyzing 31 of the protein-coding Pdx-1 target genes, we show that 29 are expressed in beta cells and, of these, 68% are down- or up-regulated in cells expressing a dominant negative mutant of Pdx-1. We additionally show that many Pdx-1 targets also interact with NeuroD1/BETA2, including the micro-RNA miR-375, a known regulator of insulin secretion.
Highlights
800,000 new cases of diabetes mellitus are diagnosed every year in the United States
For a transcription factor required for pancreatic development [6], and whose conditional deletion in mice leads to diabetes [8, 21], surprisingly little is known about Pdx-1 target genes
The ability to convert embryonic stem and liver cells into the pancreatic lineage may be facilitated by knowledge of the transcriptional and signaling networks in beta cells [52,53,54,55]
Summary
ChIP, chromatin immunoprecipitation; Dox, doxycycline; GO, gene ontology; GST, genomic signature tag; Pdx-1, pancreatic and duodenal homeobox protein-1; SACO, serial analysis of chromatin occupancy; qRT-PCR, quantitative real-time PCR; RT, reverse transcriptase; DN, dominant negative; HBSS, Hanks’ balanced salt solution; FBS, fetal bovine serum. NeuroD1 is a critical determinant of pancreatic endocrine cell differentiation [37], and both Pdx-1 and NeuroD1 regulate insulin expression [2, 38] The results from this parallel screen reveal a high degree of target overlap between these two factors and suggest that the identified binding sites may denote regulatory modules [39, 40] that are occupied by multiple factors controlling gene expression in a combinatorial fashion. One co-regulated target that we identified is the micro-RNA, miR-375, believed to negatively regulate insulin secretion [41] This result uncovers a previously unknown genetic regulatory pathway involving microRNA in pancreatic beta cells and provides a model wherein Pdx-1 and NeuroD1 control both insulin gene expression and its secretion via miR-375
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