The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.

Qiu J, Kleineidam A, Gouraud S, Tieng Yao S, Greenwood M, Hoe SZ, Hindmarch C, Murphy D

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin (AVP). Upon dehydration the SON undergoes a function-related plasticity, which includes remodelling 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 analysed 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 Electrophoretic Mobility Shift Assays (EMSA) confirmed increased binding to Stat1/Stat3, c-Myc-Max and Pbx1 sequences following 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 following dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON AVP-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation (ChIP) arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON following dehydration. These include known c-Myc targets such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1 (LAT1), ribosomal protein L24 (Rpl24), histone deactylase 2 (Hdac2), and the Ras-related nuclear GTPase (Ran).

LowCell ChIP kit

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August, 2014


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