Quantitative Assessment of Gene Targeting in Vitroand in Vivo by the Pancreatic Transcription Factor, Pdx1

Abstract

The transcription factor Pdx1 is expressed in the pancreatic ␤-cell, where it is believed to regulate several ␤-cell-specific genes. Whereas binding by Pdx1 to elements of ␤-cell genes has been demonstrated in vitro, almost none of these genes has been demonstrated to be a direct binding target for Pdx1 within cells (where complex chromatin structure exists). To determine which ␤-cell promoters are bound by Pdx1 in vivo, we performed chromatin immunoprecipitation assays using Pdx1 antiserum and chromatin from ␤-TC3 cells and Pdx1-transfected NIH3T3 cells and subsequently quantitated co-immunoprecipitated promoters using realtime PCR. We compared these in vivo findings to parallel immunoprecipitations in which Pdx1 was allowed to bind to promoter fragments in in vitro reactions. Our results show that in all cells Pdx1 binds strongly to the insulin, islet amyloid polypeptide, glucagon, Pdx1, and Pax4 promoters, whereas it does not bind to either the glucose transporter type 2 or albumin promoters. In addition, no binding by Pdx1 to the glucokinase promoter was observed in ␤-cells. In contrast, in in vitro immunoprecipitations, Pdx1 bound all promoters to an extent approximately proportional to the number of Pdx1 binding sites. Our findings suggest a critical role for chromatin structure in directing the promoter binding selectivity of Pdx1 in ␤-cells and non-␤-cells.

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…Based upon these studies, several mechanisms have been postulated: (a) increased β cell apoptosis via and Bcl-2), with resulting loss of downregulation of anti-apoptotic genes (Bcl XL functional cell mass [56,67], (b) loss of activity of key Pdx1 target genes whose products are involved in glucose-stimulated insulin transcription and secretion (including Glut2, glucokinase, MafA, Nkx6.1, insulin) [57,63,[68][69][70][71][72][73][74][75][76], and (d) loss of new β cell formation/regeneration [64,77]; in this regard, studies suggest that the action of glucagon-like peptide 1 (GLP1) in enhancing β cell growth and formation in the adult may rely upon activation of Pdx1 in β cells and potential precursor cell types, such as those residing within ducts [74,[78][79][80][81][82].…”

“…The homeodomain of Pdx1 recognizes A/T-rich DNA sequences (characteristically 5′-TAAT-3′) with affinity in the nanomolar range [120,121]. Interestingly, the range of DNA sequences bound by Pdx1 within the nucleus cannot be entirely predicted by its affinity for DNA in vitro; thus, some sequences that show preferential binding in vitro by electrophoretic mobility shift assays demonstrate little or no association within the nucleus by chromatin immunoprecipitation, and vice-versa [70,71]. At least two factors could account for this discrepancy.…”

“…These included the genes encoding insulin [68,70,71,126], Glut2 [127,128], glucokinase [129,130], islet amyloid polypeptide (IAPP) [63,75,131,132], Pax4 [133], Nkx6.1 [69,134], MafA [72], liver receptor homolog (Lrh) 1 [135], and Pdx1 itself [37]. In recent years, the direct regulation of many of these genes by Pdx1 has been confirmed using loss-of-function studies in islets and intact animals, and occupancy by Pdx1 of the endogenous genes in islets and β cell lines has been confirmed using the chromatin immunoprecipitation (ChIP) assay [70]. The insulin gene represents the best and most definitively characterized gene with respect to Pdx1 action.…”

“…Although the majority of these cofactor interactions with Pdx1 are believed to enhance the activation function of Pdx1, there is the distinct possibility that certain cofactor interactions may allow Pdx1 to aid in the repression of some genes. In this regard, the well-defined repression of genes such as glucagon [63,64,70,134] and K19 [100] by Pdx1 may reflect a repression complex formed in association with corepressor molecules.…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…Sets of PCR primers were designed to amplify a 100bp product using the Primer3 program (http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi). Fold enrichments were calculated according to (41). Briefly, since the number of molecules doubles in each cycle, fold enrichment was calculated according to the formula: 2 (dCt-dCtcontrol) .…”