We do experiments that investigate the molecular mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. These experiments are directed at determining which cis-elements in the OT &VP genes, and which transcription factors and co-regulators present in the OT &VP MCNs are responsible for the cell-specific regulation of the OT &VP genes in the SON. With respect to 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) have shown 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. Subsequent in vitro studies in our laboratory showed that the 178 and 430 bp downstream regulatory elements (REs) are interchangeable with regard to expression of the the OT &VP genes (Fields, House &Gainer, unpublished), which indicate that these REs are not responsible for the cell-specific expression. Our present experiments use AAV vectors containing deletion constructs of either the OT or VP gene promoters fused to EGFP reporters to transduce (transfect) neurons in the rat SON in vivo. After stereotaxic injection of these AAVs into rat SONs, we allow two weeks for expression of the EGFP, then perfuse fix the rat brains, and finally perform immunohistochemistry on cryostat sections of the hypothalamus to evaluate the expression of the EGFP in either the OT- or VP-MCNs. Injections of AAVs containing the 568-OT-III-EGFP-520 sequence have shown that the DNA sequence 568bp upstream of the transcription start site (TSS) in the OT gene is able to produce robust expression selectively in OT- but not VP -MCNs. Additional experiments show that AAV vectors containing 448 bp, 325bp and 216bp (but not 100bp) upstream sequences of the OT gene promoter all can support cell-specific OT gene expression in OT-MCNs (but not in VP MCNs). The 100bp upstream region does produce EGFP expression in the SON but non-selectively in the OT-and VP- MCNs, as might be expected of a core promoter region. In addition, we showed that the introns 1 and 2, and exons 2 and 3 in the OT gene are not needed for the cell-specific expression (Fields, Ponzio, Kawasaki, &Gainer, in prep). Given these data, we hypothesize that: 1) there is a repressor RE in the -216 to -100 5 upstream region of the OT gene that prevents VP cell expression, 2) 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. In the past year, we confirmed the above findings about the OT promoter, and have extended these AAV-deletion studies to examine the cis-domains in the VP promoter. AAV vectors have a limited insert capacity (4.7kb), and hence we made an initial VP construct that contained a 2 kb promoter linked via exon 1 directly to the EGFP reporter, and found that stereotaxic injection of this AAV produced robust EGFP expression only in VP-MCNs. Subsequent deletions in the VP promoter indicate that there is a powerful enhancer between 1 and 1.5kb, and that the cell-specific RE appears to reside between 288-433bp. One surprising finding in both the OT and VP deletion studies is that the mechanism of the well-studied osmotic regulation of these genes is present in all the deletion constructs, which suggests that this regulation resides in the core promoter domain, possibly at the Pol II binding site (Ponzio, Fields, Lubelski, &Gainer, in prep). Future experiments will be directed at further dissections of the -100 to -216 domain in the OT promoter and the -288 to -421 domain in the VP promoter to identify <25bp long sequences in each gene that are responsible for the cell specific expression of these genes in the SON. With such <25bp DNA sequences in hand, it should be possible to employ yeast-1-hybrid techniques to fish out and identify the transcription factors (TFs) that bind to these cis-sequences. With respect to the issue of which transcription factors and co-regulators are present &functioning in the OT &VP MCNs in the SON, we previously reported using 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 identified many specific molecules, in addition to OT and VP, that are preferentially expressed in the MCNs in the SON. However very few of these molecules were TFs. Our ability to selectively express EGFP in the OT- and VP-MCNs by the AAV strategy described above opened a novel opportunity to approach this issue. In the past year, we injected either the rAAV-p440-OT-EGFP to selectively fluorescently label OT-MCNs, or the rAAV-p2kb-VP-EGFP to selectively fluorescently label VP-MCNs, and isolated the fluorescent MCNs in the SON by laser microdissection (LCM) for molecular analyses. We then isolated RNAs from pools of such single cell dissections from each cell type and determined the relative amounts of candidate transcription factor mRNAs in the OT vs VP MCNs by qRTPCR. We accomplished this in preliminary experiments for five candidate TFs, e.g, RORA, CREB3, ARNT1 CLOCK, and AP1, and find that there is twice as much RORA in OT-MCNs vs VP MCNs. This is an important proof of principle, especially since the putative RORA activation site at -156bp upstream as previously reported for the OT gene (Chu &Zingg, 1999) is a prime candidate for the putative enhancer/repressor RE in the -216 to -100 region of the OT gene specific for OT-MCN expression. Future experiments will be done to use this LCM/ qRTPCR approach to compare the presence of putative TFs predicted by our current bioinformatic predictions of TF binding sites in the identified, relevant cis-domains in the OT and VP genes
