Functional characteristics of novel pancreatic Pax6 regulatory elements

Abstract

Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown. Using a regulatory element discovery approach, we identified two novel Pax6 pancreatic cis-regulatory elements in a poorly characterized regulatory desert. Both new elements, Pax6 pancreas cis-regulatory element 3 (PE3) and PE4, are located 50 and 100 kb upstream and interact with different parts of the Pax6 promoter and nearby non-coding RNAs. They drive expression in the developing pancreas and brain and code for multiple pancreas-related transcription factor-binding sites. PE3 binds CCCTC-binding factor (CTCF) and is marked by stem cell identity markers in embryonic stem cells, whilst a common variant located in the PE4 element affects binding of Pax4, a known pancreatic regulator, altering Pax6 gene expression. To determine the ability of these elements to regulate gene expression, synthetic transcriptional activators and repressors were targeted to PE3 and PE4, modulating Pax6 gene expression, as well as influencing neighbouring genes and long non-coding RNAs, implicating the Pax6 locus in pancreas function and diabetes.

Polymer Simulations of Heteromorphic Chromatin Predict the 3D Folding of Complex Genomic Loci

“…To develop this strategy, we investigated the folding of 5 Mb around the Pax6 locus using three different immortalized cell lines that expressed Pax6 at different levels ( Figure S1 ), referred to as Pax6-OFF, ON, and HIGH cell lines. Pax6 is flanked by two constitutively expressed housekeeping genes, with enhancer elements within the Pax6 gene itself and at regions ∼50 kb upstream and ∼95 kb downstream; these are referred to below as the up- and downstream regulatory regions (URR and DRR, respectively) ( Buckle et al., 2018 , Kleinjan et al., 2006 ). The histone modification H3K27ac ( Figure S1 A), usually associated with enhancers and transcriptional activation, was found at the gene and distal enhancers when Pax6 was active, and these regions at enhancers broadened significantly in HIGH-activity cells.…”

“…We also recently combined the TF model with the popular loop extrusion (LE) model for chromosome organization ( Pereira et al., 2018 ), which explains features of chromatin loops mediated by cohesin and CTCF ( Fudenberg et al., 2016 , Sanborn et al., 2015 ). While this strategy successfully predicts large-scale features of genome organization, we show below that it cannot accurately predict the folding of the complex Pax6 genomic locus at high resolution, which we probed experimentally at different levels using fluorescence in situ hybridization (FISH) imaging and Capture-C. Pax6 is surrounded by constitutively expressed genes and multiple enhancers, providing a paradigm for complex genetic interactions ( Buckle et al., 2018 , Lacomme et al., 2018 , McBride et al., 2011 ).…”

Heterogeneous loop model to infer 3D chromosome structures from Hi-C

“…The DRR contains several DNase Ihypersensitive sites and the SIMO enhancer, which was identified in transgenic reporter gene studies of developing mouse embryos 71,72 . Another cis-regulatory element PE3 within URR has recently been identified from mouse pancreatic β-cells (β-TC3) 70 .…”

Polymer Simulations of Heteromorphic Chromatin Predict the 3-D Folding of Complex Genomic Loci

“…S1), referred to as Pax6-OFF, ON and HIGH cell lines. Pax6 is flanked by two constitutively expressed housekeeping genes, with enhancer elements within the Pax6 gene itself, and at regions ~50 kb upstream and ~95 kb downstream -these are referred to below as the up and downstream regulatory regions (URR and DRR respectively 11,19 ). The histone modification H3K27ac ( Fig.…”