title: The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.
abstract: The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.
authors.affiliation: School of Clinical Sciences (J.Q., A.K., S.G., S.T.Y., M.G., C.H., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; and Department of Physiology (S.Z.H., C.H., D.M.), Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.
authors.forename: Jing
authors.initials: J
authors.lastname: Qiu
authors.forename: Anna
authors.initials: A
authors.lastname: Kleineidam
authors.forename: Sabine
authors.initials: S
authors.lastname: Gouraud
authors.forename: Song Tieng
authors.initials: ST
authors.lastname: Yao
authors.forename: Mingkwan
authors.initials: M
authors.lastname: Greenwood
authors.forename: See Ziau
authors.initials: SZ
authors.lastname: Hoe
authors.forename: Charles
authors.initials: C
authors.lastname: Hindmarch
authors.forename: David
authors.initials: D
authors.lastname: Murphy
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003681:Dehydration / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D007031:Hypothalamus / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D046228:Microarray Analysis / Q000379:methods*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000378:metabolism; D059467:Transcriptome
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA