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. 2001 Dec 4;98(25):14481-6.
doi: 10.1073/pnas.241349398. Epub 2001 Nov 20.

A transcription factor regulatory circuit in differentiated pancreatic cells

S F Boj  1 , M Parrizas, M A Maestro, J Ferrer
Affiliations

A transcription factor regulatory circuit in differentiated pancreatic cells

S F Boj et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the human genes encoding hepatocyte nuclear factors (HNF) 1alpha, 1beta, 4alpha, and IPF1(PDX1/IDX1/STF1) result in pancreatic beta cell dysfunction and diabetes mellitus. In hepatocytes, hnf4alpha controls the transcription of hnf1alpha, suggesting that this same interaction may operate in beta cells and thus account for the common diabetic phenotype. We show that, in pancreatic islet and exocrine cells, hnf4alpha expression unexpectedly depends on hnf1alpha. This effect is tissue-specific and mediated through direct occupation by hnf1alpha of an alternate promoter located 45.6 kb from the previously characterized hnf4alpha promoter. Hnf1alpha also exerts direct control of pancreatic-specific expression of hnf4gamma and hnf3gamma. Hnf1alpha dependence of hnf4alpha, hnf4gamma, hnf3gamma, and two previously characterized distal targets (glut2 and pklr) is established only after differentiated cells arise during pancreatic embryonic development. These studies define an unexpected hierarchical regulatory relationship between two genes involved in human monogenic diabetes in the cells, which are relevant to its pathophysiology. Furthermore, they indicate that hnf1alpha is an essential component of a transcription factor circuit whose role may be to maintain differentiated functions of pancreatic cells.

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Figures

Figure 1
Figure 1
Pancreatic-specific hnf1α dependence of hnf4α. RT-PCR analysis of hnf4α mRNA (encompassing exons 2 and 3) in purified tissues of hnf1α−/− and hnf1α+/+ mice. β-actin, tbp, or hprt was coamplified as internal control. Arrowheads indicate expected position of hnf4α PCR products.
Figure 2
Figure 2
Pancreatic hnf4α mRNA is transcribed from a tissue-specific alternate promoter. (A) Alignment of human and mouse genomic sequences encoding the 5′ end of pancreatic hnf4α mRNA (exon 1D) and its 5′ flanking region; ↓, 5′ end of human pancreatic islet RNA rapid amplification of cDNA ends product; ▾ and ▿, prediction of transcription initiation by using tssw and nppw programs (19); ⧫, 5′ end of mouse hnf4α7 cDNA (21); and ●, exon-intron boundary. A horizontal line indicates the predicted initiator codon. The hnf1-binding site is boxed. (B) RT-PCR analysis of hnf4α transcripts containing either exon 1A or exon 1D sequences in tissues from hnf1α−/− and hnf1α+/+ mice. Arrowheads indicate expected position of hnf4α PCR products. (C) RT-PCR analysis of hnf4α mRNA in pancreatic endocrine (endo) and exocrine (exo) fractions from hnf1α−/− and hnf1α+/+ mice. Glut2 and amylase mRNA were assayed to indicate tissue purity. (D) Schematic indicating the genomic position of exon 1D and P2 promoter relative to the known (P1) HNF4α promoter. (E) ChIP analysis of acetylated histones H3 and H4 (AcH3 and AcH4), indicating tissue-specific, hnf1α-dependent hyperacetylation of P2 in islets. The gapdh promoter represents a control hyperacetylated chromatin region, which is unaffected by hnf1α. PI, preimmune serum. Input 1:50–600, diluted DNA purified before ChIP to indicate expected results in the absence of enrichment of specific DNA fragments.
Figure 3
Figure 3
Hnf1α occupies hnf4α P2 promoter DNA in vitro and in vivo. (A, lanes 1–9) Electrophoretic mobility-shift assay showing interaction of a pancreatic nuclear complex to an oligonucleotide containing the site boxed in Fig. 2A. Binding is blocked by excess (×2–×200) unlabeled probe (P2) but not by an oligonucleotide with two single base substitutions (P2m). Anti-hnf1α Ab but not preimmune (PI) serum supershifts the complex (arrowhead). Analogous results are observed by using synthetic hnf1α (lanes 11–15), whereas no retardation is seen with an in vitro translation reaction performed with a control plasmid (lane 10). Similar results are seen with gluthathione S-transferase hnf1α, purified islet, and mouse hepatocyte nuclear extracts (not shown). (B) ChIP assays indicate that hnf1α occupies the endogenous hnf4α P2 promoter in islets (lane 1, compare to preimmune serum in lane 2 and input DNA), whereas in liver it contacts the P1 promoter (lane 5). Input 1:50–300, diluted input DNA.
Figure 4
Figure 4
Hnf1α dependence of hnf4γ and hnf3γ in pancreatic cells (A) RT-PCR analysis of hnf4γ and hnf3γ mRNA in hnf1α−/− and hnf1α+/+ mice. Arrowheads indicate expected position of hnf PCR products. (B) RT-PCR analysis of hnf4γ and hnf3γ mRNA in pancreatic endocrine and exocrine tissues from hnf1α−/− and hnf1α+/+ mice (see Fig. 2C for more details and tissue-specific markers). (C) ChIP analysis indicating that hnf1α directly occupies the hnf4γ promoter and hnf3γ enhancer in vivo in pancreatic islets. See Figs. 2D and 3B and text for further details. (D) ChIP analysis indicating that islet hnf4γ promoter chromatin is enriched in acetylated H4 and depends on hnf1α function. See also legend of Fig. 2E.
Figure 5
Figure 5
Ontogeny of the pancreatic hnf1α-dependent genetic program. (A and B) RT-PCR analysis of dissected pancreatic tissue at indicated embryonic ages reveals that hnf1α dependence of hnf4α (P2), hnf4γ, hnf3γ, glut2, and pklr is established in parallel with the surge of differentiated pancreatic cells at ≈E15. (C) Immunofluorescence analysis of glut2 (green) in timed embryonic pancreas. Loss of glut2 in hnf1α−/− embryos is first elicited at E15.5 in some insulin-positive cells (red) (arrowheads). At E18.5 all insulin-positive cells lack glut2 in hnf1α−/− embryos.
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