RNA profiling and chromatin immunoprecipitation-sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors

Abstract

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.

Fig 1

Experimental flow charts and starting material. (A) For RNA profiling, the E10.5 pancreatic bud epithelia were mechanically dissected, and RNA was extracted from three pools of three buds from each genotype. The RNA was twice linearly amplified and then reverse transcribed, and RNA probes were generated for hybridization in triplicate to Affymetrix GeneChip Mouse Genome 430 2.0. (B) Whole-mount immunocytochemistry for PDX1 showing high PDX1 expression in the dorsal (dp) and ventral (vp) pancreatic buds at E10.5 (upper panels). Expression is lower in the PTF1a KO mouse. Expression between the buds is in the duodenum (bottom), and the antral stomach (top) serves as a reference for expression unaffected by PTF1a. The bottom panels show a decrease in PDX1 levels at E11.5 in the WT and a collapse of dorsal pancreatic bud in the KO. All pictures were captured with the same exposure time. Scale bar, 200 μm. (C) For ChIP, 266-6 cells were cross-linked, sheared, and immunoprecipitated with anti-PTF1a or control anti-IgG antibodies. Immunoprecipitated chromatin and control input were sequenced at high throughput. A subset of potential binding sites was confirmed by QPCR comparing chromatin precipitated by anti-PTF1a and anti-IgG antibodies.

Fig 2

Distribution of Ptf1a ChIP-Seq peaks in transcriptionally regulated versus nonregulated genes. The x axis shows the base distance from the most upstream transcription start site, noted as 0. The y axis is the number of peaks of more than 20 tags per 10-kb interval. The red line shows that for genes that depend on PTF1a for their expression in pancreas progenitors, binding peaks are enriched from kb −50 to kb +100 around the start site compared to genes repressed by PTF1a (green line) or random genes (blue line) which had no peak enrichment.

Fig 3

Four pancreatic progenitor transcription factors are direct PTF1a targets. The top panels show double immunohistochemistry with antibodies directed against PTF1a (A to D) and either MNX1 (A′), PDX1 (B′), NKX6.1 (C′) or OC1 (D′) on WT pancreas at E10.5. The same section is shown in A and A′, B and B′, C and C′, D and D′. Scale bar = 50 μm. ∗denotes unspecific staining in gut lumen. (E to H) Decrease in expression detected by qRT-PCR in E10.5 Ptf1a^−/− compared to Ptf1a^+/− and Ptf1a^+/+ for each of the 4 transcription factors. n = 3 to 4 pools of 3 buds for each genotype. Statistical significance was tested by nonparametric Mann-Whitney test. ∗ P < 0.05. Scale bar = 200 μm for B and C. (I to L) Peaks of sequences enriched upon PTF1a ChIP. The ChIP-Seq peaks are aligned with the UCSC genome browser v37 on the gene loci, and the conservation of the region among vertebrates is aligned. The color-coded primers used to independently validate the bound regions by QPCR are shown with arrowheads below the gene structure. The pink boxes show areas of PTF1a binding. For each gene, a control unbound region in the locus is shown to demonstrate the specificity of peak binding as well as anti-IgG ChIP control.

Fig 4

PTF1a is a major Mnx1 activator through a distant enhancer. (A) Immunohistochemistry with anti-PDX1 (green) and anti-MNX1 (red) antibodies at E9.5 and E10.5. The same exposure time was used for Ptf1a^+/+, ptf1a^+/−, and ptf1a^−/− littermates and reveals reduced MNX1 expression in the dorsal bud of Ptf1a^+/− at E10.5. The reduction is even more drastic in KO dorsal (dp) and ventral (vp) pancreatic buds. Insets show MNX1 only in the dorsal pancreas. Scale bar = 50 μm. (B) A map of the 11-kb reporter construct is aligned with conserved sequences. (C) The 11-kb reporter introduced in transgenic mice recapitulates expression in motor neurons and hindgut but not in the pancreas. Scale bar in C and E = 100 μm. (D) A map of the 212-kb BAC reporter construct is aligned with conserved sequences. (E) The 212-kb BAC reporter introduced in transgenic mice recapitulates expression in motor neurons (mn) and hindgut as well as in the entire dorsal gut tube, including the pancreas. The posterior foregut is shown as an inset, including stomach (st), dorsal (dp), and ventral (vp) pancreas. (F) Luciferase reporter constructs in which the Mnx1 minimal promoter (1.4 kb) was associated or not with distant enhancer elements. X marks a mutation introduced in the PTF1a consensus sequence of the kb −51.8 enhancer. (G) Relative luciferase activity (RLU) in a transcriptional activity assay in which these constructs are transfected into 266-6 cells. The kb −51.8 enhancer increases activity and loses this ability upon mutation of the PTF1a consensus element. Statistical significance was tested by nonparametric Mann-Whitney test. ∗∗, P < 0.005.

Fig 5

New PTF1a targets. (A) In situ hybridization with RhoV, P2rx1, BMP7, or Nr5a2 probes (blue) in E10.5 Ptf1a^+/+ and Ptf1a^−/− pancreata. Scale bar = 200 μm. This reveals a strong decrease of expression in Ptf1a KO. The pancreatic bud is systematically identified on an adjacent section stained with anti-Pdx1. These genes are also expressed in terminal end buds which contain exocrine cells expressing PTF1a at E14.5. (B) The four boxes show the peaks of sequences enriched upon PTF1a ChIP-Seq. The peaks are aligned with the UCSC genome browser on the gene loci, and the conservation of the region among vertebrates is aligned. One or multiple PTF1a binding peaks are revealed in the vicinity of each of these genes.

Fig 6

Transcription factor network which maintains pancreas progenitors.