LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function

Abstract

We have determined the cistrome and transcriptome for the nuclear receptor liver receptor homolog-1 (LRH-1) in exocrine pancreas. Chromatin immunoprecipitation (ChIP)-seq and RNA-seq analyses reveal that LRH-1 directly induces expression of genes encoding digestive enzymes and secretory and mitochondrial proteins. LRH-1 cooperates with the pancreas transcription factor 1-L complex (PTF1-L) in regulating exocrine pancreas-specific gene expression. Elimination of LRH-1 in adult mice reduced the concentration of several lipases and proteases in pancreatic fluid and impaired pancreatic fluid secretion in response to cholecystokinin. Thus, LRH-1 is a key regulator of the exocrine pancreas-specific transcriptional network required for the production and secretion of pancreatic fluid.

Figure 1.

Identification of LRH-1 target genes in exocrine pancreas by RNA-seq and ChIP-seq analyses. (A) Immunoblots of LRH-1 protein (top panel, n = 2 mice per genotype) and qPCR of LRH-1 mRNA (bottom panel, n = 4 per group) from pancreas of Flox and Lrh1^t−/− mice. (NS) Nonspecific band. (B) Comparison and overlap of LRH-1 target genes. RNA-seq identified genes up-regulated or down-regulated twofold or more in Lrh1^t−/− pancreas compared with Flox pancreas. ChIP-seq analysis from pancreas of wild-type mice identified genes with at least one LRH-1-binding site within 50 kb of a gene. The overlap of genes found in both data sets containing a consensus LRHRE are shown. (C,D) Distribution of LRH-1 ChIP-seq peaks across the genome (C) and relative to gene transcriptional start sites (TSSs) (D). (E) Analysis of LRH-1 ChIP-seq peak sequences revealed the consensus LRHRE as an overrepresented motif. Numbers of LRHRE-positive peaks and associated genes are shown. (F) Direct LRH-1 target genes identified by RNA-seq and ChIP-seq that were up-regulated or down-regulated in the Lrh1^t−/− pancreas are shown.

Figure 2.

LRH-1 regulates exocrine pancreas-specific genes. (A) LRH-1-regulated genes identified by RNA-seq were confirmed by qPCR by comparing expression in Flox and Lrh1^t−/− (KO) pancreas (n = 5 per group). (B) ChIP-seq data were confirmed by conventional ChIP using an LRH-1 antibody and Flox and Lrh1^t−/− pancreas (n = 3 per group). (Cel) Carboxyl ester lipase; (Pnlip) pancreatic lipase; (Ela1) elastase 1; (Ctrl) chymotrypsin-like; (Cpa1) carboxypeptidase A1; (Shp) small heterodimer partner; (Aqp12) aquaporin 12; (Sycn) syncollin; (Gatm) glycine amidinotransferase; (Gls2) glutaminase 2.

Figure 3.

LRH-1 and PTF1-L coregulate exocrine pancreas-specific genes. (A) ChIP-seq and RNA-seq data from Lrh1^t−/−, E17.5 Rbpjl^−/−, and control mouse pancreas were used to identify genes that contained overlapping LRH-1 and PTF-1L ChIP-seq peaks (within 50 kb) and that were dysregulated twofold or more in the same direction (either up or down) in the absence of LRH-1 or PTF1-L. The relative mRNA levels in the Lrh1^t−/− and Rbpjl^−/− mice compared with control mice are shown. Common LRH-1/PTF1-L target genes are listed according to function. (B) 293T cells were cotransfected with expression plasmids for RBPJL, LRH-1, and PTF1A as indicated together with luciferase reporter plasmids containing the mouse Rbpjl, Cel, Cpa2, and Ela1 promoters, or these same promoters in which either the LRHRE or E-boxes were mutated. Schematics showing the relative positions of LRHREs, E-boxes, and TC-boxes in each promoter are shown. (C) 293T cells were cotransfected with the Ela1-luc reporter plasmid and expression plasmids for RBPJL, LRH-1, and PTF1A^W298A as indicated. In B and C, n = 3 ± SEM. Luciferase activity is normalized to vector alone. (D) Coimmunoprecipitation experiments were performed with either whole pancreas extracts from Flox and Lrh1^t−/− mice (left panels) or 293T cells transfected with Flag-tagged LRH-1 in the presence or absence of PTF1A (right panel). Immunoprecipitations were done with a PTF1A antibody, and immunoblotting was done with an LRH-1 antibody (top left panel) or an anti-Flag antibody (top right panel). Immunoblots for total input LRH-1 and PTF1A are shown at the bottom.

Figure 4.

LRH-1 regulates the secretion and composition of pancreatic fluid. (A) Pancreatic fluid volumes from basal and CCK-stimulated secretions were collected for 10 min each, and volumes were normalized to body weight (n = 5–7 mice per group). (B) Pancreatic secretions from A were resolved by SDS-PAGE and visualized by silver staining. Each lane represents pancreatic fluid pooled from two mice. Expected positions of abundant digestive enzymes are indicated. (CEL) Carboxyl ester lipase; (AMY2) amylase 2; (PNLIP) pancreatic lipase; (CPA2) carboxypeptidase A2; (KLK1) kallikrein 1; (ELA1) elastase 1; (CTRL) chymotrypsin-like. (C) CCK-stimulated pancreatic secretions from three Flox and three Lrh1^t−/− mice were analyzed by Western analysis for PNLIP, CPA2, KLK1, and ELA1.