Islet-1 regulates Arx transcription during pancreatic islet alpha-cell development

Abstract

Aristaless related homeodomain protein (Arx) specifies the formation of the pancreatic islet α-cell during development. This cell type produces glucagon, a major counteracting hormone to insulin in regulating glucose homeostasis in adults. However, little is known about the factors that regulate Arx transcription in the pancreas. In this study, we showed that the number of Arx(+) cells was significantly reduced in the pancreata of embryos deficient for the Islet-1 (Isl-1) transcription factor, which was also supported by the reduction in Arx mRNA levels. Chromatin immunoprecipitation analysis localized Isl-1 activator binding sites within two highly conserved noncoding regulatory regions (Re) in the Arx locus, termed Re1 (+5.6 to +6.1 kb) and Re2 (+23.6 to +24 kb). Using cell line-based transfection assays, we demonstrated that a Re1- and Re2-driven reporter was selectively activated in islet α-cells, a process mediated by Isl-1 in overexpression, knockdown, and site-directed mutation experiments. Moreover, Arx mRNA levels were up-regulated in islet α-cells upon Isl-1 overexpression in vivo. Isl-1 represents the first known activator of Arx transcription in α-cells, here established to be acting through the conserved Re1 and Re2 control domains.

FIGURE 1.

The number of Arx⁺ cells is reduced in the Pdx1-Cre;Isl- 1^LoxP/LoxP embryos. A–D, immunostaining with glucagon and Arx antibodies of E15.5 pancreatic sections from control and Pdx1-Cre;Isl-1^LoxP/LoxP embryos. E, quantitative analysis of Arx⁺ cell populations as normalized to Pax6⁺ cells in the neighboring section. n = 6 for control and n = 5 for mutants. Data represents the mean ± S.E. *, p value <0.05.

FIGURE 2.

Arx expression is elevated Pdx1^PB-Isl-1-Myc mice. A, diagram representing the Pdx1^PB-Isl-1-Myc transgenic construct, which contains the pdx1^PB fragment of the pdx1 gene and the hsp68 minimal promoter. B, Western blot by α-Isl-1 and α-Myc analysis of total cell lysate from islets of 8-week-old control and Pdx1^PB-Isl-1-Myc mice. The asterisk denotes a nonspecific band. C, Isl-1 mRNA levels in Pdx1^PB-Isl-1-Myc and littermate control mice. Results are presented as mean ± S.E. *, p <0.05. D–G, immunohistochemical analysis for glucagon or insulin and Myc expression in 8-week-old Pdx1^PB-Isl-1-Myc and control mice. H, Arx mRNA levels in Pdx1^PB-Isl-1-Myc and control mice. Results are presented as mean ± S.E. *, p <0.05. I–L, costaining of Arx with glucagon or insulin in 8-week-old Pdx1^PB-Isl-1-Myc and control mice. Insets show the magnified views of the outlined areas. The asterisk denotes autofluorescence from red blood cells.

FIGURE 3.

Isl-1 binds to Re1 and Re2 of the mouse Arx gene. A, schematic diagram of the Arx locus illustrating the locations of Re1 (orange box, +5.6 to +6.1) and Re2 (green box, +23.6 to +24 kb). TSS, transcription start site; Ex, exon. B, the ChIP-Seq image depicts Isl-1 occupancy within Arx Re1 (chrX:89544959–89545500) and Re2 (chrX:89563002–89563440) in αTC1–6 cells. The conservation between mouse and other species within this region was shown using the University of California Santa Cruz genome browser. C and D, Re1 and Re2 binding to Isl-1 was readily detected in adult mouse islets and αTC1–6 cells by standard ChIP but to a lesser extent in βTC3 cells. Results were normalized to Isl-1 binding to the PEPCK promoter. Data are presented as the mean ± S.E. n = 3; p value <0.05.

FIGURE 4.

Re1 and Re2 mediate Arx transcription in αTC1–6 cells. A, pGL4.27-Re1, pGL4.27-Re2, and pGL4.27-Re1/2 were transfected into αTC1–6 cells in the presence or absence of a Isl-1-Myc expression plasmid. B, Western blot analysis using Isl-1 and control α-tubulin antibodies demonstrates a ∼70% reduction of Isl-1 protein levels in Isl-1 shRNA lentivirus-treated αTC1–6 cells. C, endogenous Arx mRNA expression was down-regulated relative to HPRT in Isl-1 shRNA treated αTC1–6 cells. D, Isl-1 shRNA knockdown reduced pGL4.27-Re1 and pGL4.27-Re2 reporter activity in αTC1–6 cells. Results are presented as the mean ± S.E. *, p <0.05. pGL4.27-Arx activity was normalized to that of the pRL-SV40 Renilla luciferase in A and D.

FIGURE 5.

The Isl-1 binding sites in Re1 and Re2 are necessary for αTC1–6 cell activity. Sequence identity within Arx Re1 (A) and Arx Re2 (B) between mouse, human, and rat. Core Isl-1 binding site sequences are shown in bold, and the lines above demarcate the EMSA probe. Nucleotide mutated for luciferase reporter (C) and EMSA (Fig. 6C) assays are labeled in red. Nonconserved nucleotides are labeled in green. C, the activity of wild-type and Isl-1 binding site mutants (red) of pGL4.27-Re1 and pGL4.27-Re2 in αTC1–6 cells. The pRL-SV40 normalized data were presented as the mean ± S.E. *, p <0.05.

FIGURE 6.

Isl-1 binding sites in Re1 and Re2. A and B, as a screen of putative Isl-1 binding elements, the radiolabeled MafA-R3 Isl-1 site probe (17) was used in reactions with in vitro translated Isl-1-Myc and MafA-R3, Re1, or Re2 competitor oligonucleotides. Isl-1 and Myc-epitope antibodies were used to localize the Isl-1-Myc:MafA complex. Ab, antibody; IVT, in vitro translation; Neg, without Isl-1-Myc; Pos, with Isl-1-Myc. C, Re2–2, Re2–3, and Re2–6 probes were incubated with αTC1–6 nuclear extract. The specificity of Isl-1:Arx-Re2 binding was determined by Isl-1 antibody addition and competition with excess of unlabeled wild-type (WT) and Isl-1 binding site mutant (MT).