We are addressing two key issues and associated hypotheses related to molecular mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. The first issue is to evaluate which cis-elements in the OT &VP genes are responsible for the cell-specific regulation. Previous transgenic studies (Young et al,1990;Jeong et al, 2001;Davies et al, 2003) &in vitro analyses using biolistics and organotypic cultures (Fields et al, 2003) show that the expression of OT &VP is due to the coordinate action of specific cis-elements found 554 bp 5upstream of the transcription start site (TSS) in the OT gene, and <3.4 kbp 5 upstream of the TSS in the VP gene, and 178, and 430 bp 3downstream of exon 3 in the VP and OT genes, respectively. Recent in vitro studies showed that the 178 and 430 bp downstream regulatory elements (REs) are interchangeable with regard to expression of the the OT &VP genes (Fields, unpublished), indicating that these REs are not responsible for the cell-specific expression. More recent experiments using AAV vectors to transduce (transfect) neurons in the rat SON in vivo with the 563-OT-III-EGFP-520 sequence have confirmed that the DNA sequence 563bp upstream of the TSS in the OT gene is able to produce robust expression selectively in OT magnocellular neurons (MCNs) (Fields &Ponzio, unpublished). Additional experiments have indicated that AAV vectors containing 448 bp, 325bp and 216bp upstream sequences of the OT gene can support cell specific OT gene expression (vs VP cell expression). However, the 216 bp upstream region also leads to expression in a non-MCN population of neurons dorsal to the SON. In contrast, the 100bp upstream region produces clear expression but with no specificity at all, as might be expected of a core promoter region. Given these data, we hypothesize that specific cis regulatory elements (REs) located in the 325bp 5upstream region in the OT gene, are critical for its cell-specific and regulated expression in OT magnocellular neurons (MCNs) in the SON. More specifically, we hypothesize: 1) that there is a repressor RE in the -216 to -100 region of the OT gene preventing VP cell expression, 2) ) that there is an enhancer RE in the -216 to -100 region of the OT gene specific for OT cell expression, 3) that there is another repressor RE in the -325 to -216 region of the OT gene which prevents expression in the supra SON (non-MCN) population of neurons, and 4) there may be additional enhancer REs in the -440 to -216 region of the OT gene specific for the OT cell expression. Finally, the putative ROR alpha activation site at -180 to -160bp upstream previously described in the OT gene (Chu &Zingg, 1999) is a candidate for the putative enhancer RE in the -216 to -100 region of the OT gene specific for OT cell expression. Experiments in progress that are testing these assertions are: 1) Determine if the minimal REs (325 vs 216) essential for basal cell specific expression in the SON will also support regulated expression during acute and chronic osmotic stimulation. 2) Determine if the minimal REs (325 vs 216) essential for basal cell specific expression in the SON will also work in the PVN &SCN. 3) Do more deletion experiments actual &in silico to further dissect the putative REs in regions -325 to -216 and -216 to -100. (It might be possible to test whether there is an additional OT enhancer in -440 to -216 and only one VP repressor, i.e, in -216 to 100bp, by removing -216 to -100 from p440bp and seeing if the resulting sequence without the -216 to -100bp region is robust in both the OT &VP cells,etc.) 5) Begin deletion analyses of the 3.4kbp 5 regions in the VP gene to determine which REs are critical for cell-specific and regulated expression in the SON. The second issue is to determine which transcription factors and co-regulators are present &functioning in the OT &VP MCNs in the SON With respect to this issue, we previously used differential analyses of single cell OT &VP expression libraries (Yamashita et al, 2002) and laser microdissection of the SON and microarrays (Mutsuga et al, 2004;2005;Yue et al, 2006),and we identified a number of specific molecules, in addition to OT and VP, that are preferentially expressed in the MCNs in the SON, and which are substantially regulated in expression during osmotic adaptation. We hypothesize that cell specific expression of the OT &VP and other genes in the MCNs will depend on specific proteins in the transcriptosome (e.g, transcription factors, enhancers,suppressors, coactivators, cosuppressors , etc) in each cell type. Experiments in progress that address this hypothesis are: 1) To examine whether specific candidate transcription factors, enhancers, suppressors, coactivators, cosuppressors genes are in fact present in the SON, and if they are selectively expressed in OT vs VP MCNs. One approach will be to perform laser microdissection of the SON, isolation of its RNAs and to determine the presence of candidate transcription factor mRNAs in the SON by qRTPCR. Some of the candidate transcription factors we will test as regulators of OT &VP transcription in the SON are CREB, ER beta, GR, and RORalpha. The strategy will be to use lentiviral and/or AAV vector transduction to express the wildtype genes or to knockdown their expression (using shRNA, or dominant negative specific genes) in the SON in vivo, and to then measure changes in the OT &VP hnRNAs by qRTPCR.

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