Identification of the Downstream Targets of SIM1 and ARNT2, a Pair of Transcription Factors Essential for Neuroendocrine Cell Differentiation

SIM1 and ARNT2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors that control the differentiation of neuroendocrine lineages in the mouse hypothalamus. Heterozygous Sim1 mice also develop early onset obesity, possibly due to hypodevelopment of the hypothalamus. Although SIM1 and ARNT2 form heterodimers to direct the same molecular pathway, knowledge of this pathway is limited. To facilitate the identification of their downstream genes, we combined an inducible gene expression system in a neuronal cell line with microarray analysis to screen for their transcriptional targets. This method identified 268 potential target genes of SIM1/ARNT2 that displayed >1.7-fold induced expression. 15 of these genes were subjected to Northern analysis, and a high percentage of them were confirmed to be up-regulated. In vivo, several of these genes showed neuroendocrine hypothalamic expression correlating with that of Sim1. Furthermore, we found that expression of two of these potential targets, the Jak2 and thyroid hormone receptor beta2 genes, was lost in the neuroendocrine hypothalamus of the Sim1 mutant. The expression and predicted functions of many of these genes provide new insight into both the Sim1/Arnt2 action in neuroendocrine hypothalamus development and the molecular basis for the Sim1 haplo-insufficient obesity phenotype. FIG1"> WIDTH=86 HEIGHT=200 SRC="/small/bc4538389001.gif" ALT=" "> View larger version (25K): FIG1">[in this window] FIG1" onClick="startTarget('FIG1', 340, 640); this.href='FIG1'" onMouseOver="window.status='View figure in a separate window'; return true" TARGET="FIG1">[in a new window] FIG. 1. A, schematic representation of the plasmids used in this study: pTRE2hyg, empty vector without a cDNA insert used as a control; pTRE-SF, pTRE2hyg harboring full-length Sim1 and Arnt2 joined by the IRES; pTRE-SN-Gal4, pTRE2hyg harboring the Sim1 N-terminal bHLH/PAS domain (SimN) fused to the Gal4 activation domain, IRES, and Arnt2; pTRE-SN-VP16, pTRE2hyg harboring SimN fused to the VP16 activation domain, IRES, and Arnt2; pTRE-Arnt2 and pTRESim-VP16, pTRE2hyg harboring Arnt2 and SimN fused to VP16, respectively; pTet-On, for expression of the tetracycline-controlled transactivator rtTA; pTet-tTS, for expression of the tetracycline-controlled silencer tTS; pML/6C-WT (17), a luciferase reporter driven by the adenovirus major late promoter linked to four copies of CME (4xCME) for SIM1/ARNT2 binding; pML/6C-AM (17), a similar reporter linked to four copies of CME with point mutations (CMEm) as shown. TRE, tetracycline-responsive element for rTA and tTS binding; PCMV, cytomegalovirus-derived enhancer/promoter for high level transcription; PminCMV, minimal cytomegalovirus-derived enhancer/promoter. B, comparison of the activities of SIM1 variants in the Tet-On system. Transient transfections of the Neuro-2a cells were carried out using the plasmids indicated. The fold induction was calculated as relative luciferase activities of the doxycycline-treated (Dox+) over the mock-treated (Dox-) samples. The relative luciferase activities were normalized to the beta-galactosidase activity from cotransfected pSV-betagal (data not shown). Results represent the mean of three independent experiments, and the error bars are the S.D. values. C, activation of the reporter by the SimN-VP16 fusion gene depends on Arnt2 expression from either a separate plasmid or a single plasmid linked by IRES and the wild-type CME, but not the mutant CME. Transfections, luciferase measurement, and normalization were the same as described for B. Arbitrary luciferase values were used to indicate CME activation. 10.1074/jbc.M304489200