FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2003149404> ?p ?o ?g. }

• W2003149404 endingPage "44867" @default.
• W2003149404 startingPage "44857" @default.
• W2003149404 abstract "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 β2 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. 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 β2 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. The neuroendocrine hypothalamus mediates homeostasis by regulating peptidergic hormone secretion of the pituitary. Discrete hypothalamic secretory neurons mediate this function. These neurons include the oxytocin (OT) 1The abbreviations used are: OT, oxytocin; VP, vasopressin; CRH, corticotropin-releasing hormone; TRH, thyrotropin-releasing hormone; bHLH, basic helix-loop-helix; PVN, paraventricular nucleus; SON, supraoptic nucleus; CME, central nervous system midline enhancer; IRES, internal ribosomal entry site; Dox, doxycycline; MC3R, melanocortin-3 receptor; TRβ2, thyroid hormone receptor β2; IL-6Rα, interleukin-6 receptor α; ISH, in situ hybridization; E18.5, embryonic day 18.5; NLOT, nucleus of the lateral olfactory track; AVN, anteroventral nucleus; rtTA, reverse tetracycline-controlled transactivator; tTS, tetracycline transcriptional silencer; bHLH, basic helix-loop-helix.-, vasopressin (VP)-, corticotropin-releasing hormone (CRH)-, thyrotropin-releasing hormone (TRH)-, and somatostatin-producing neurons. OT and VP neurons project to the posterior pituitary, where they release hormones directly into the bloodstream, whereas the CRH, TRH, and somatostatin neurons project to the median eminence, which in turn carries their secreted hormones to the anterior pituitary to modulate pituitary secretion (1Kandel E.R. Shwartz J.H. Jessell T.M. Principle of Neuroscience. 3rd ed. Appleton & Lange, East Norwalk, CT1991Google Scholar). Despite extensive studies of the physiological functions of these hormones, the molecular pathways directing their expression in specific cell lineages are less well known. Analyses of Sim1 and Arnt2 mutant mice have demonstrated that these transcription factors are essential for the terminal differentiation of the aforementioned neurons (2Michaud J.L. Rosenquist T. May N.R. Fan C.-M. Genes Dev. 1998; 12: 3264-3275Crossref PubMed Scopus (299) Google Scholar, 3Michaud J.L. DeRossi C. May N.R. Holdener B.C. Fan C.-M. Mech. Dev. 2000; 90: 253-261Crossref PubMed Scopus (146) Google Scholar, 4Hosoya T. Oda Y. Takahashi S. Morita M. Kawauchi S. Ema M. Yamamoto M. Fujii-Kuriyama Y. Genes Cells. 2001; 6: 361-374Crossref PubMed Scopus (91) Google Scholar, 5Keith B. Adelman D.M. Simon M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6692-6697Crossref PubMed Scopus (126) Google Scholar). In the absence of either gene, the precursors of these neurons are born normally, but fail to form the anatomical neuroendocrine centers, i.e. the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) in the anterior hypothalamus, and do not produce any of the hormones (2Michaud J.L. Rosenquist T. May N.R. Fan C.-M. Genes Dev. 1998; 12: 3264-3275Crossref PubMed Scopus (299) Google Scholar, 3Michaud J.L. DeRossi C. May N.R. Holdener B.C. Fan C.-M. Mech. Dev. 2000; 90: 253-261Crossref PubMed Scopus (146) Google Scholar, 4Hosoya T. Oda Y. Takahashi S. Morita M. Kawauchi S. Ema M. Yamamoto M. Fujii-Kuriyama Y. Genes Cells. 2001; 6: 361-374Crossref PubMed Scopus (91) Google Scholar, 5Keith B. Adelman D.M. Simon M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6692-6697Crossref PubMed Scopus (126) Google Scholar). The collective loss of these neuroendocrine hormones may cause the observed perinatal lethality of the Sim1 and Arnt2 mutants (2Michaud J.L. Rosenquist T. May N.R. Fan C.-M. Genes Dev. 1998; 12: 3264-3275Crossref PubMed Scopus (299) Google Scholar, 3Michaud J.L. DeRossi C. May N.R. Holdener B.C. Fan C.-M. Mech. Dev. 2000; 90: 253-261Crossref PubMed Scopus (146) Google Scholar, 4Hosoya T. Oda Y. Takahashi S. Morita M. Kawauchi S. Ema M. Yamamoto M. Fujii-Kuriyama Y. Genes Cells. 2001; 6: 361-374Crossref PubMed Scopus (91) Google Scholar, 5Keith B. Adelman D.M. Simon M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6692-6697Crossref PubMed Scopus (126) Google Scholar). Intriguingly, heterozygous Sim1 mice develop early onset obesity, proposed to be due to hypodevelopment of the neuroendocrine hypothalamus (6Michaud J.L. Boucher F. Melnyk A. Gauthier F. Goshu E. Levy E. Mitchell G.A. Himms-Hagen J. Fan C.-M. Hum. Mol. Genet. 2001; 10: 1465-1473Crossref PubMed Scopus (235) Google Scholar). A balanced chromosomal translocation disrupting SIM1 (7Holder Jr., J.L. Butte N.F. Zinn A.R. Hum. Mol. Genet. 2000; 9: 101-108Crossref PubMed Google Scholar) and a haploid interstitial deletion of chromosome 6 encompassing SIM1 (8Faivre L. Cormier-Daire V. Lapierre J.M. Colleaux L. Jacquemont S. Genevieve D. Saunier P. Munnich A. Turleau C. Romana S. Prieur M. De Blois M.C. Vekemans M. J. Med. Genet. 2002; 39: 594-596Crossref PubMed Scopus (91) Google Scholar) have also been shown to be associated with profound obesity in humans. Sim1 and Arnt2 are homologs of Drosophila sim and tango, respectively (9Ema M. Morita M. Ikawa S. Tanaka M. Matsuda Y. Gotoh O. Saijoh Y. Fujii H. Hamada H. Kikuchi Y. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 5865-5875Crossref PubMed Scopus (136) Google Scholar, 10Fan C.-M. Kuwana E. Bulfone A. Fletcher C.F. Copeland N.G. Jenkins N.A. Crews S. Martinez S. Puelles L. Rubenstein J.L. Tessier-Lavigne M. Mol. Cell. Neurosci. 1996; 7: 1-16Crossref PubMed Scopus (137) Google Scholar, 11Franks R.G. Crews S.T. Mech. Dev. 1994; 45: 269-277Crossref PubMed Scopus (33) Google Scholar, 12Hirose K. Morita M. Ema M. Mimura J. Hamada H. Fujii H. Saijo Y. Gotoh O. Sogawa K. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 1706-1713Crossref PubMed Scopus (222) Google Scholar, 13Sonnenfeld M. Ward M. Nystrom G. Mosher J. Stahl S. Crews S. Development. 1997; 124: 4571-4582PubMed Google Scholar, 14Crews S.T. Fan C.-M. Curr. Opin. Genet. Dev. 1999; 9: 580-587Crossref PubMed Scopus (159) Google Scholar). These genes belong to the family of basic helix-loop-helix (bHLH)/PAS (Per-Arnt-Sim) domain-containing proteins, many of which are important regulators of development and physiology (14Crews S.T. Fan C.-M. Curr. Opin. Genet. Dev. 1999; 9: 580-587Crossref PubMed Scopus (159) Google Scholar). DNA binding assays in vitro have demonstrated that SIM1 and ARNT2 form heterodimers and bind the core sequence TACGTC, named central nervous system midline enhancer (CME) (3Michaud J.L. DeRossi C. May N.R. Holdener B.C. Fan C.-M. Mech. Dev. 2000; 90: 253-261Crossref PubMed Scopus (146) Google Scholar, 12Hirose K. Morita M. Ema M. Mimura J. Hamada H. Fujii H. Saijo Y. Gotoh O. Sogawa K. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 1706-1713Crossref PubMed Scopus (222) Google Scholar, 14Crews S.T. Fan C.-M. Curr. Opin. Genet. Dev. 1999; 9: 580-587Crossref PubMed Scopus (159) Google Scholar, 15Wharton Jr., K.A. Franks R.G. Kasai Y. Crews S.T. Development. 1994; 120: 3563-3569Crossref PubMed Google Scholar, 16Kasai Y. Stahl S. Crews S. Gene Expr. 1998; 7: 171-189PubMed Google Scholar). The CME was originally identified in the enhancer regions of sim/tango downstream genes (13Sonnenfeld M. Ward M. Nystrom G. Mosher J. Stahl S. Crews S. Development. 1997; 124: 4571-4582PubMed Google Scholar, 15Wharton Jr., K.A. Franks R.G. Kasai Y. Crews S.T. Development. 1994; 120: 3563-3569Crossref PubMed Google Scholar). Multimerized CME can mediate sim/tango-dependent central nervous system midline expression in the fly (11Franks R.G. Crews S.T. Mech. Dev. 1994; 45: 269-277Crossref PubMed Scopus (33) Google Scholar, 13Sonnenfeld M. Ward M. Nystrom G. Mosher J. Stahl S. Crews S. Development. 1997; 124: 4571-4582PubMed Google Scholar, 14Crews S.T. Fan C.-M. Curr. Opin. Genet. Dev. 1999; 9: 580-587Crossref PubMed Scopus (159) Google Scholar, 15Wharton Jr., K.A. Franks R.G. Kasai Y. Crews S.T. Development. 1994; 120: 3563-3569Crossref PubMed Google Scholar, 16Kasai Y. Stahl S. Crews S. Gene Expr. 1998; 7: 171-189PubMed Google Scholar). When linked to a minimal adenovirus major late promoter-driven reporter gene, CME can also mediate SIM1/ARNT2-dependent transcriptional activation of the reporter in cultured mammalian cells, albeit weakly (17Moffett P. Pelletier J. FEBS Lett. 2000; 466: 80-86Crossref PubMed Scopus (43) Google Scholar). Deletion analyses of SIM1 and ARNT2 demonstrate that their basic domains are required for CME recognition, their bHLH/PAS domains for heterodimerization, and their C termini for transcriptional regulation (9Ema M. Morita M. Ikawa S. Tanaka M. Matsuda Y. Gotoh O. Saijoh Y. Fujii H. Hamada H. Kikuchi Y. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 5865-5875Crossref PubMed Scopus (136) Google Scholar, 12Hirose K. Morita M. Ema M. Mimura J. Hamada H. Fujii H. Saijo Y. Gotoh O. Sogawa K. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 1706-1713Crossref PubMed Scopus (222) Google Scholar, 17Moffett P. Pelletier J. FEBS Lett. 2000; 466: 80-86Crossref PubMed Scopus (43) Google Scholar, 18Probst M.R. Fan C.-M. Tessier-Lavigne M. Hankinson O. J. Biol. Chem. 1997; 272: 4451-4457Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, when the SIM1 C terminus is fused to the Gal4 DNA-binding domain and tested in a different cell line using a Gal4-thymidine kinase promoter-driven reporter, it acts as a repressor (9Ema M. Morita M. Ikawa S. Tanaka M. Matsuda Y. Gotoh O. Saijoh Y. Fujii H. Hamada H. Kikuchi Y. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 5865-5875Crossref PubMed Scopus (136) Google Scholar). These results suggest that SIM1 can act as a repressor or an activator depending on the context of the reporter assay. Brn2, a POU domain-encoding gene, is a downstream target of SIM1/ARNT2 in vivo (2Michaud J.L. Rosenquist T. May N.R. Fan C.-M. Genes Dev. 1998; 12: 3264-3275Crossref PubMed Scopus (299) Google Scholar, 3Michaud J.L. DeRossi C. May N.R. Holdener B.C. Fan C.-M. Mech. Dev. 2000; 90: 253-261Crossref PubMed Scopus (146) Google Scholar, 4Hosoya T. Oda Y. Takahashi S. Morita M. Kawauchi S. Ema M. Yamamoto M. Fujii-Kuriyama Y. Genes Cells. 2001; 6: 361-374Crossref PubMed Scopus (91) Google Scholar, 5Keith B. Adelman D.M. Simon M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6692-6697Crossref PubMed Scopus (126) Google Scholar). In both Sim1 and Arnt2 mutants, Brn2 expression in the prospective neuroendocrine cells is lost. Furthermore, Brn2 mutant mice have a selective defect in CRH-, VP-, and OT-expressing neurons (19Burbach J.P. Luckman S.M. Murphy D. Gainer H. Physiol. Rev. 2001; 81: 1197-1267Crossref PubMed Scopus (291) Google Scholar, 20Nakai S. Kawano H. Yudate T. Nishi M. Kuno J. Nagata A. Jishage K. Hamada H. Fujii H. Kawamura K. Shiba K. Noda T. Genes Dev. 1995; 9: 3109-3121Crossref PubMed Scopus (235) Google Scholar, 21Schonemann M.D. Ryan A.K. McEvilly R.J. O'Connell S.M. Arias C.A. Kalla K.A. Li P. Sawchenko P.E. Rosenfeld M.G. Genes Dev. 1995; 9: 3122-3135Crossref PubMed Scopus (250) Google Scholar), which is a subset of the Sim1 and Arnt2 mutant defects. BRN2 has also been shown to bind to the CRH promoter and to activate its transcription (22Li P. He X. Gerrero M.R. Mok M. Aggarwal A. Rosenfeld M.G. Genes Dev. 1993; 7: 2483-2496Crossref PubMed Scopus (150) Google Scholar, 23Ramkumar T. Adler S. Mol. Endocrinol. 1999; 13: 1237-1248Crossref PubMed Google Scholar). The genes employed by SIM1/ARNT2 to specify the other neuroendocrine hormone gene expression in distinctive cell types remain unexplored. To study the molecular pathways by which Sim1 and Arnt2 control the development of the hypothalamic secretory neurons and mediate energy homeostasis, we combined an inducible gene expression system with microarray analysis to screen for their downstream targets. Below, we describe the genes identified by this screen and the resulting implications for the Sim1/Arnt2-operated molecular pathway. Plasmids—The SIM1 N-terminal bHLH/PAS domain (1044 bp from ATG to an internal EcoRV, referred to as SIMN) was fused in-frame with Gal4 or VP16 activation domains. These fusion forms of Sim1 and full-length Sim1 cDNAs were cloned into a pIRES vector (Clontech) with the Arnt2 cDNA inserted 3′ to the internal ribosomal entry site (IRES) to make Sim1-IRES-Arnt2, SimN-VP16-IRES-Arnt2, and SimN-Gal4-IRES-Arnt2 cassettes. These cassettes were cloned into the pTRE2hyg vector (Clontech). In addition, Sim1, Arnt2, and SimN-VP16 were individually cloned into pTRE2hyg to make pTRE-Sim1, pTREArnt2, and pTRE-Sim-VP16, respectively. pTet-On and pTet-tTS (Clontech) were used for expression of the tetracycline-regulated activator rtTA and repressor tTS, respectively, to render doxycycline (Dox)-dependent regulation of pTRE-driven expression of cloned cDNAs. The CME-driven luciferase reporters pML/6C-WT and pML/6C-AM were gifts from Dr. J. Pelletier (17Moffett P. Pelletier J. FEBS Lett. 2000; 466: 80-86Crossref PubMed Scopus (43) Google Scholar). 50 ng of pSV-βgal (Invitrogen) was included in all transfections, and the β-galactosidase activity was measured (LacZ assay kit, Promega) for normalization. Cell Culture—The Neuro-2a cells (American Type Culture Collection, Manassas, VA) were cultured in Eagle's minimal essential medium (Vitacell, American Type Culture Collection) and 10% bovine serum. FuGENE 6 reagent (Roche Applied Science) was used for DNA transfection. For transient transfections, each plasmid was used at 250 ng in a final 1 μg of total DNA for each well of a 6-well dish (Falcon). The plasmids used for each transfection are indicated in the figures. For a stable cell line, Neuro-2a cells were transfected with 10 μg of pTet-On and selected with 200 μg/ml G418 (Sigma) to obtain individual clones. Selected colonies were propagated and transfected with pTREhyg-Luc (Clontech) to test their Dox (1.5 μg/ml; Clontech) responsiveness by assaying inducible luciferase activity. The clone with the lowest background was transfected with 2 μg of pTRE-SN-VP16 and 10 μg of pTet-tTS and selected with 150 μg/ml hygromycin (Roche Applied Science) to obtain secondary clones. Individual clones were then tested for their Dox-regulated SIMN-VP16/ARNT2 activity by assaying for pML/6C-WT reporter activity under mock and Dox treatment conditions. Clone 37 was chosen for microarray study. Luciferase activities were measured by luciferin (Sigma) light emission using Monolight 2010 (Analytical Luminescence Laboratory). Microarray Hybridization and Data Analysis—Total RNA was isolated using TRIzol reagent (Invitrogen), followed by the QIAGEN RNeasy method. Microarray hybridization using the MG-U74v2 A gene chip was performed using a service provided by Neurologic Functional Genomics. Triplicate hybridizations with independently synthesized probes were conducted using the same batch of RNA isolated from the untreated and Dox-treated clone 37 cell lines and the parental cell line. Hybridization signals were normalized by Affymetrix Suite software. The data sets were filtered by absent and present calls using the Affymetrix Datamining tool, i.e. genes that displayed inconsistent signals within the same oligonucleotide probe set are excluded. Based on the general background of the data, genes displayed signals <30 arbitrary units under both mock and Dox treatment conditions (after normalization) were excluded for further analyses. These remaining genes were subjected to Student's t test, with p ≤ 0.05 considered significant. We arbitrarily chose a 1.7-fold increase as a cutoff threshold for selecting genes for further investigation based on Northern confirmation rate of genes displaying various fold inductions. Northern and Western Analyses—10 μg of total RNA was resolved on 1% agarose gels and transferred to GeneScreen membrane (PerkinElmer Life Sciences) for Northern hybridization. For the melanocortin-3 receptor (MC3R) and Tbr1 genes, fragments were amplified by reverse transcription-PCR using total RNA isolated from newborn mouse brain. The primers used were 5′-ggcaacctgcactctc-3′ and 5′-catgcccaagttcatgc-3′ for the MC3R gene and 5′-gacaacctggagagaag-3′ and 5′-aactggttttgtgcc-3′ for Tbr1. For other genes, IMAGE clones were obtained from American Type Culture Collection and ResGen: Tlx2 (clone 935644), Jak2 (clone 1391934), Mtpn (clone 5579590), thyroid hormone receptor β2 (TRβ2; clone 1600024), Chrne (clone 5127017), Grin1 (clone UI-M-API-agn-g-11-0-UI), Naca (clone 2136152), and interleukin-6 receptor α (IL-6Rα; clone 1463277). Each clone was sequenced to confirm authenticity. The glyceraldehyde-3-phosphate dehydrogenase probe is a PCR fragment obtained from Clontech. The DNA fragment of each gene was labeled with [α-32P]dCTP by random priming (Stratagene) and used for hybridization according to the protocol provided by PerkinElmer Life Sciences for using the GeneScreen membrane. Cell lysates were resolved on a 4∼15% gradient gel (Bio-Rad); transferred to a Hybond membrane (Amersham Biosciences); and probed with antibodies against the VP16 activation domain (Clontech), ARNT2 (Santa Cruz Biotechnology), and γ-tubulin (Sigma). Horseradish peroxidase-conjugated secondary antibodies (Sigma) coupled with the chemiluminescence reaction (Amersham Biosciences) were used to visualize these proteins. In Situ Hybridization (ISH)—The brains of CD1 mice at embryonic day 18.5 (E18.5; vaginal plug date is designated as embryonic day 0.5) were snap-frozen in OCT compound and cryosectioned at 20-μm thickness. In Fig. 6, Sim1 heterozygotes in a BL6 backcrossed background were mated to obtain E18.5 brains from Sim1 mutant and wild-type siblings for ISH. Digoxigenin-labeled sense and antisense probes of the genes specified in the figures were synthesized using SP6, T7, or T3 polymerase and used at ∼1 μg/ml for hybridization of the brain sections according to Schaeren-Wuemers and Gerfin-Moser (24Schaeren-Wuemers N. Gerfin-Moser A. Histochemistry. 1993; 100: 431-440Crossref PubMed Scopus (1085) Google Scholar). Inducible Expression of SIM1 and ARNT2 in the Neuro-2a Cell Line—Overexpression of transcription factors should lead to changes in the expression levels of their downstream target genes. To achieve controlled SIM1/ARNT2 overexpression, we chose to use the Tet-On inducible system (see “Experimental Procedures” for details). In this system, pTet-On and pTet-tTS (Fig. 1A) are used to confer tetracycline-dependent regulation of pTRE (tetracycline-responsive element)-driven genes, in this case, Sim1 and Arnt2 (Fig. 1A; diagrammed in Fig. 3A).Fig. 3Microarray analysis of SIMN-VP16/ARNT2-up-regulated genes in clone 37 cells. A, experimental schemes of microarray screen for SimN-VP16/Arnt2 downstream targets using clone 37 cells. In the absence of Dox (Dox-; mock-induced), the tetracycline-controlled transcriptional silencer tTS bound to the tetracycline-responsive element (TRE) and repressed the transcription of SimN-VP16-IRES-Arnt2. In the presence of Dox (black dot, Dox+; Dox-induced), tTS dissociated from the tetracycline-responsive element, whereas the tetracycline-controlled transactivator rtTA bound to the tetracycline-responsive element and activated the transcription of SimN-VP16-IRES-Arnt2. Total RNA from each condition was isolated, labeled, and hybridized to microarrays, and the data were compared for differentially expressed genes under the two conditions. B, scatter plot of the entire set of gene expression data from the microarray. Each dot represents one gene. The normalized average signal intensity under the mock and Dox treatment conditions are shown on the x and y axes in arbitrary fluorescence values. Gray lines indicate the 1.7-, 2-, 10-, and 20-fold differential expression thresholds. The locations of three genes are marked: Arnt2, Jak2, and TRβ2. Up-regulation of Arnt2 is an indicator that the Dox-treated sample did contain induced SimN-VP16-IRES-Arnt2 transcripts. C, table illustrating the distribution of the 268 up-regulated genes into seven categories. The transcription regulators represent genes that are known to participate in regulating transcription directly. The signaling component category includes secreted peptide factors, membrane receptors, and signaling effectors. Metabolic enzymes include enzymes in known metabolic pathways. Channels and transporters include neurotransmitter receptors or transporters for small molecules. The cell adhesion and migration category includes adhesion molecules, extracellular matrix proteins, and guidance molecules for cell migration and axonal projection. The miscellaneous group includes genes with documented functions not overlapping with the above categories, e.g. immunity-related proteins. The uncharacterized genes are expressed sequence tags in the data base whose functions have not been investigated. The functional categories and the number of genes within each category are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We reasoned to implement this system in a neuronal cell line, as we are interested in the function of Sim1/Arnt2 in the hypothalamus. Because the SIM1 C terminus has been reported to repress or activate transcription in different contexts (9Ema M. Morita M. Ikawa S. Tanaka M. Matsuda Y. Gotoh O. Saijoh Y. Fujii H. Hamada H. Kikuchi Y. Fujii-Kuriyama Y. Mol. Cell. Biol. 1996; 16: 5865-5875Crossref PubMed Scopus (136) Google Scholar, 17Moffett P. Pelletier J. FEBS Lett. 2000; 466: 80-86Crossref PubMed Scopus (43) Google Scholar), we surveyed the activity of SIM1/ARNT2 in various neuronal cell lines using the CME-driven luciferase reporter (pML/6C-WT) assay devised by Moffett and Pelletier (17Moffett P. Pelletier J. FEBS Lett. 2000; 466: 80-86Crossref PubMed Scopus (43) Google Scholar). SIM1 and ARNT2 together activated this reporter expression in NB41A3, N1E-115, and Neuro-2a cells in a CME-dependent manner (data not shown). The Neuro-2a cells were chosen for further study due to their homogeneous morphology and high transfection efficiency. Under the various Dox treatment conditions tested, we observed an optimal ∼4-fold activation of the pML/6C-WT reporter by SIM1 and ARNT2 in Neuro-2a cells (Fig. 1B), regardless of whether they were expressed from a single plasmid linked by the IRES or from separate plasmids (data not shown). Concerned that their activity might be too low to activate endogenous genes for our assay, we constructed a potent SIM1 fusion activator. Sim1 hybrid constructs with the SIM1 C terminus replaced by the Gal4 and VP16 (SIMN-VP16) activation domains were cloned into the pTRE2hyg vectors pTRE-SNGal4 and pTRE-SN-VP16, respectively (Fig. 1A). Arnt2 was placed downstream of the IRES located 3′ to these Sim1 variants for coexpression (Fig. 1A). Plasmids carrying these Sim1 variants as well as Arnt2 were transfected into Neuro-2a cells to compare their Dox-regulated proficiency in activating the pML/6C-WT reporter. Upon Dox treatment, pTRE-SN-VP16 conferred a 27-fold activation of the reporter compared with the 12- and 4-fold reporter activation rendered by pTRE-SN-Gal4 and pTRE-SF, respectively (Fig. 1B). SIMN-VP16 appears to function with similar specificity as SIM1, as it also required ARNT2 and wild-type CME sites for reporter gene activation (Fig. 1C). pTRE-SN-VP16 was therefore used to establish a stable Neuro-2a clonal cell line for Dox-inducible expression of SIMNVP16 and ARNT2 (see “Experimental Procedures” for details). One clone (clone 37) with this characteristic was obtained. As shown in Fig. 2A, this clone expressed readily detectable levels of the SimN-VP16-IRES-Arnt2 transcript of the predicted size of 4.2 kb upon Dox treatment. The SIMN-VP16 and ARNT2 proteins were also detected (Fig. 2B). No SimN-VP16-IRES-Arnt2 transcripts or SIMN-VP16 and ARNT2 proteins were found under the mock-induced conditions (Fig. 2, A and B). For the temporally regulated transcription activity of SIMN-VP16/ARNT2 in the clone 37 cells, we determined the time course of pML/6C-WT reporter activity upon Dox treatment. After 8, 16, and 24 h of Dox treatment, the achieved induction of the reporter was ∼6-, 160-, and 530-fold, respectively (Fig. 2C). Therefore, SIMN-VP16/ARNT2 transcription activity can be induced to high levels between 16 and 24 h of Dox treatment in the clone 37 cells. Genes Regulated by SIMN-VP16/ARNT2 in the Microarray Analysis—We prepared total RNA from mock- and Dox-treated clone 37 cells in parallel. Northern analysis was used to determine induction of the SimN-VP16-IRES-Arnt2 transcript prior to subjecting the RNA to microarray analysis. Fig. 3A outlines the experimental flowchart. The Affymetrix mouse chip MGU74v2 A (containing 12,000 probe sets) was used. Independent probe syntheses from the same batch of RNA and hybridizations were performed in triplicate. The data sets were normalized, analyzed, and presented as a scatter correlation plot in Fig. 3B (see “Experimental Procedures” for a detailed description). The presence of the Arnt2 gene on this chip serves as an internal control of Dox induction. A total of 268 genes displaying >1.7-fold (arbitrarily chosen) increased expression were considered significantly up-regulated by t test, with p ≤ 0.05. These 268 genes can be divided into several functional categories as summarized in Fig. 3C and described in the legend thereof. The identity of each gene and its fold induction are listed in Table I. Importantly, no significant gene expression changes were found in the parental cell lines treated with Dox versus the mock-treated clone 37 cell line, indicating that the up-regulated genes in the Dox-treated clone 37 sample are regulated by SIMN-VP16/ARNT2.Table IUp-regulated genes from microarray analysisGenBank™/EBI accession no.Gene name and descriptionChangefoldTranscription regulatorsU89489LIM domain binding 2 (Ldb2)1.74AW125812SRY box-containing gene 10 (Sox10)1.78Y08361Reversion-induced LIM gene (Ril)1.81M58566Zinc finger protein 36, C3H type-like 1 (Zfp36l1)1.81AF087035Musculin (Msc)1.81AB025922GLI-Krüppel family member GLI1 (Gli1)1.82U36576Nuclear factor of activated T-cells, cytoplasmic 2 (Nfatc2)1.82AI931748CCR4-NOT transcription complex, subunit 7 (Cnot7)1.83AV214633POU domain, class 5, transcription factor 1 (Pou5f1)1.85D13664Osteoblast-specific factor 2 (Osf2)1.88AA864065Core-binding factor-β (Cbfb)1.93D49658LIM homeobox protein 8 (Lhx8)1.93AV369921Early growth response 1 (Egr1)2.01U61110Eyes absent 1 homolog (Drosophila) (Eya1)2.02M75953T-cell leukemia homeobox 2 (Tlx2)2.11L38622Transcription regulator, SIN3 yeast homolog B (Sin3b)2.12AJ223069Transcription factor 3 (Tcf3)2.12AF007110Transformed mouse 3T3 cell double minute 4 (Mdm4)2.14AV320880Nuclear receptor subfamily 5, A1 (Nr5a1)2.15Z35294Mature T-cell proliferation 1 (MTCP-1)2.18AF040242Nuclear antigen Sp1002.19U90538Forkhead box B1 (Foxh1)2.3Z36885Member of ETS oncogene family (ELK4)2.36U36799Retinoblastoma-like 2 (Rbl2)2.4AI956211Nascent polypeptide-associated complex α-polypeptide (Naca)2.43L09600Nuclear factor, erythroid-derived 2 (Nfe2)2.48AI843911Glucocorticoid-induced leucine zipper (Gilz)2.51AV377670GATA-binding protein 2 (Gata2)2.56U91840Factor in the germline α (Figla)2.58X13945Lung carcinoma myc-related oncogene 12.71AB008192MAD homolog 3, Drosophila (Smad3, Dr...)2.73AW210248General transcription factor III A (Gtf3α)2.77U15548Thyroid hormone receptor β2 (Trebeta-2)2." @default.
• W2003149404 created "2016-06-24" @default.
• W2003149404 creator A5037762357 @default.
• W2003149404 creator A5044174169 @default.
• W2003149404 creator A5051566104 @default.
• W2003149404 creator A5068376812 @default.
• W2003149404 date "2003-11-01" @default.
• W2003149404 modified "2023-10-15" @default.
• W2003149404 title "Identification of the Downstream Targets of SIM1 and ARNT2, a Pair of Transcription Factors Essential for Neuroendocrine Cell Differentiation" @default.
• W2003149404 cites W1546146845 @default.
• W2003149404 cites W1807487415 @default.
• W2003149404 cites W1965359283 @default.
• W2003149404 cites W1966170752 @default.
• W2003149404 cites W1971108334 @default.
• W2003149404 cites W1974442900 @default.
• W2003149404 cites W1982957409 @default.
• W2003149404 cites W2005131002 @default.
• W2003149404 cites W2005687249 @default.
• W2003149404 cites W2010840481 @default.
• W2003149404 cites W2026106328 @default.
• W2003149404 cites W2026644592 @default.
• W2003149404 cites W2029117845 @default.
• W2003149404 cites W2031605069 @default.
• W2003149404 cites W2040901874 @default.
• W2003149404 cites W2042289885 @default.
• W2003149404 cites W2050442786 @default.
• W2003149404 cites W2053074173 @default.
• W2003149404 cites W2067844666 @default.
• W2003149404 cites W2070582535 @default.
• W2003149404 cites W2079949985 @default.
• W2003149404 cites W2081677113 @default.
• W2003149404 cites W2083092470 @default.
• W2003149404 cites W2085682237 @default.
• W2003149404 cites W2087482176 @default.
• W2003149404 cites W2092027349 @default.
• W2003149404 cites W2094611185 @default.
• W2003149404 cites W2098181391 @default.
• W2003149404 cites W2099616263 @default.
• W2003149404 cites W2141166900 @default.
• W2003149404 cites W2144392815 @default.
• W2003149404 cites W2159527243 @default.
• W2003149404 cites W2161615619 @default.
• W2003149404 cites W2165953630 @default.
• W2003149404 cites W2181813824 @default.
• W2003149404 cites W2185816512 @default.
• W2003149404 cites W2186630782 @default.
• W2003149404 cites W4297692503 @default.
• W2003149404 doi "https://doi.org/10.1074/jbc.m304489200" @default.
• W2003149404 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12947113" @default.
• W2003149404 hasPublicationYear "2003" @default.
• W2003149404 type Work @default.
• W2003149404 sameAs 2003149404 @default.
• W2003149404 citedByCount "41" @default.
• W2003149404 countsByYear W20031494042012 @default.
• W2003149404 countsByYear W20031494042013 @default.
• W2003149404 countsByYear W20031494042014 @default.
• W2003149404 countsByYear W20031494042017 @default.
• W2003149404 countsByYear W20031494042018 @default.
• W2003149404 countsByYear W20031494042020 @default.
• W2003149404 countsByYear W20031494042021 @default.
• W2003149404 crossrefType "journal-article" @default.
• W2003149404 hasAuthorship W2003149404A5037762357 @default.
• W2003149404 hasAuthorship W2003149404A5044174169 @default.
• W2003149404 hasAuthorship W2003149404A5051566104 @default.
• W2003149404 hasAuthorship W2003149404A5068376812 @default.
• W2003149404 hasBestOaLocation W20031494041 @default.
• W2003149404 hasConcept C104317684 @default.
• W2003149404 hasConcept C116834253 @default.
• W2003149404 hasConcept C127413603 @default.
• W2003149404 hasConcept C138885662 @default.
• W2003149404 hasConcept C1491633281 @default.
• W2003149404 hasConcept C179926584 @default.
• W2003149404 hasConcept C185592680 @default.
• W2003149404 hasConcept C18903297 @default.
• W2003149404 hasConcept C21547014 @default.
• W2003149404 hasConcept C2776207758 @default.
• W2003149404 hasConcept C41895202 @default.
• W2003149404 hasConcept C54355233 @default.
• W2003149404 hasConcept C86339819 @default.
• W2003149404 hasConcept C86803240 @default.
• W2003149404 hasConcept C95444343 @default.
• W2003149404 hasConceptScore W2003149404C104317684 @default.
• W2003149404 hasConceptScore W2003149404C116834253 @default.
• W2003149404 hasConceptScore W2003149404C127413603 @default.
• W2003149404 hasConceptScore W2003149404C138885662 @default.
• W2003149404 hasConceptScore W2003149404C1491633281 @default.
• W2003149404 hasConceptScore W2003149404C179926584 @default.
• W2003149404 hasConceptScore W2003149404C185592680 @default.
• W2003149404 hasConceptScore W2003149404C18903297 @default.
• W2003149404 hasConceptScore W2003149404C21547014 @default.
• W2003149404 hasConceptScore W2003149404C2776207758 @default.
• W2003149404 hasConceptScore W2003149404C41895202 @default.
• W2003149404 hasConceptScore W2003149404C54355233 @default.
• W2003149404 hasConceptScore W2003149404C86339819 @default.
• W2003149404 hasConceptScore W2003149404C86803240 @default.
• W2003149404 hasConceptScore W2003149404C95444343 @default.
• W2003149404 hasIssue "45" @default.
• W2003149404 hasLocation W20031494041 @default.

• next