Pdx1 and BETA2/NeuroD1 Participate in a Transcriptional Complex That Mediates Short-range DNA Looping at the Insulin Gene

Abstract

The activity of the insulin gene, Ins, in islet ␤ cells is thought to arise in part from the synergistic action of the transcription factors Pdx1 and BETA2/NeuroD1. We asked how the binding of these factors to A and E elements many tens or hundreds of base pairs upstream of the start site could influence activity of transcriptional machinery. We therefore tested the hypothesis that the complex of Pdx1 and BETA2/NeuroD1 maintains a DNA conformation such that distal regions of the gene are brought into proximity of the promoter and coding region. We show by coimmunoprecipitation that Pdx1 and BETA2/ NeuroD1 exist within a complex and that the two physically interact with one another in this complex as assessed by fluorescence resonance energy transfer. Consistent with this interaction, we found that the two factors simultaneously occupy the same fragment of the Ins gene in ␤ cell lines using the chromatin immunoprecipitation/re-chromatin immunoprecipitation assay. Using a modification of the chromosome conformation capture assay in vitro and in ␤ cells, we observed that Pdx1 and BETA2/NeuroD1 mediate looping of a segment of Ins that brings EcoRI sites located at ؊623 and ؉761 bp (relative to the transcriptional start site) in proximity to one another. This looping appears to be dependent in vitro upon an intact A3 binding element, but not upon the E2 element. Based on our findings, we propose a model whereby Pdx1 and BETA2/ NeuroD1 physically interact to form a nucleoprotein complex on the Ins gene that mediates formation of a short DNA loop. Our results suggest that such short loop conformations may be a general mechanism to permit interactions between transcription factors and basal transcriptional machinery.

Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells

“…Long range looping by the 3C assay between distant regulatory elements has also been documented for other genes, including the C-reactive protein (37), the peptidylarginine deaminase 3 (38,39), and interferon ␥ genes (40). More recently, similar to our results with chromatin looping for the HO-1 gene, DNA looping over short ranges (ϳ100 bp to 10 kb) have been described for the murineinducible nitric-oxide synthase (41,42), human CD68 (43), insulin (44), and the neonatal Fc receptor (FcRn) for IgG genes (45). In the case of the FcRn gene, DNA looping interactions were reported between intronic NF-B sequences and the FcRn promoter spanning over a ϳ12-kb distance (45).…”

A dynamic regulatory network explains ParaHox gene control of gut patterning in the sea urchin

“…Likewise, three transcription factors of the Forkhead family (FoxY, FoxD and FoxP), mainly expressed in foregut and midgut territories, were significantly downregulated, thus suggesting their potential involvement in Xlox gut patterning control. NeuroD1, a regulator of insulin production in vertebrates (Babu et al, 2008;Kaneto et al, 2009), was also strongly downregulated, indicating a possible evolutionary conserved cooperative function for Xlox and NeuroD1 in the sea urchin larva. Moreover, a GABA receptor and a cholinergic receptor were highly downregulated and we associated these results with the potential role of Xlox in the specification of some neural cells of the ciliary band from a group of ectodermal cells in which Xlox transcripts have been revealed ).…”

Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

“…In vitro 3C assays were performed essentially as previously described with minor modifications (Babu et al, 2008;Inoue and Negishi, 2009;Li et al, 2010). Nuclear extracts (100 mg) prepared from HepG2 cells transfected with empty vector or cFos expression vector were incubated with 50 ng linearized CYP2C9 plasmid (containing 23077 to 11 of the CYP2C9 promoter) at room temperature for 45 minutes.…”