Regulation of Oxytocin and Vasopressin Gene Expression in the hypothalamus.

我们做了实验，研究调节下丘脑细胞特异性OT和VP基因表达的分子机制。这些实验旨在确定OT和VP基因中的哪些顺式元件，以及OT和VP mcn中存在的哪些转录因子和共调节因子负责SON中OT和VP基因的细胞特异性调节。关于OT和VP基因中的哪些顺式元件负责细胞特异性调控，以往的转基因研究(Young et al,1990; Jeong et al, 2001；Davies et al ., 2003)和利用生物学和器官型培养的体外分析（Fields et al ., 2003）表明，OT和VP的表达是由于特定顺式元件的协同作用，这些顺式元件在OT基因的转录起始位点（TSS）上游554 bp 5，在VP基因的TSS上游< 3.4 kbp 5，在VP和OT基因的外显子3下游178 bp 3和430 bp 3。我们实验室随后的体外研究表明，178 bp和430 bp下游调控元件（REs）在OT和VP基因的表达中是可互换的（Fields, House & Gainer，未发表），这表明这些REs不负责细胞特异性表达。我们目前的实验使用含有OT或VP基因启动子缺失结构的AAV载体与EGFP报告基因融合，在大鼠SON体内转导（转染）神经元。将这些aav立体定向注射到大鼠SONs中后，我们允许EGFP表达两周，然后灌注固定大鼠脑，最后在下丘脑低温切片上进行免疫组织化学，以评估OT-或VP-MCNs中EGFP的表达。注射含有568-OT-III-EGFP-520序列的aav表明，OT基因转录起始位点（TSS）上游568bp的DNA序列能够在OT-而不是VP - mcns中选择性地产生稳健的表达。进一步的实验表明，含有448 bp、325bp和216bp（但不是100bp）上游OT基因启动子序列的AAV载体都可以支持OT-MCNs中细胞特异性的OT基因表达（但不支持VP MCNs）。上游100bp区域在SON中确实产生EGFP表达，但在ot -和VP- MCNs中非选择性地产生EGFP表达，这可能是核心启动子区域的预期。此外，我们发现OT基因的内含子1和2以及外显子2和3对于细胞特异性表达是不需要的（Fields, Ponzio, Kawasaki, & Gainer, In prep）。根据这些数据，我们假设：1)有一个阻遏再保险在-216 -100 5的上游区域无法阻止VP细胞基因表达,2)有一个增强再保险在-216 - -100区域OT基因的特定的细胞不表达,3),还有一个阻遏再保险在-325 - -216区域的OT基因从而防止表达上的儿子(non-MCN)人口的神经元,和4)可能有额外的增强物在-440 - -216区域OT基因特定的细胞表达。在过去的一年里，我们证实了上述关于OT启动子的发现，并将这些aav缺失研究扩展到VP启动子的顺式结构域。AAV载体的插入容量有限（4.7kb），因此我们制作了一个包含2kb启动子的初始VP结构，该启动子通过外显子1直接连接到EGFP报告基因，并发现该AAV的立体定向注射仅在VP- mcn中产生稳健的EGFP表达。随后VP启动子的缺失表明，在1 - 1.5kb之间存在一个强大的增强子，并且细胞特异性RE似乎位于288-433bp之间。在OT和VP缺失研究中，一个令人惊讶的发现是，这些基因的渗透调控机制在所有缺失构建中都存在，这表明这种调控存在于核心启动子区域，可能在Pol II结合位点（Ponzio, Fields, Lubelski, & Gainer, in prep）。未来的实验将针对OT启动子的-100至-216结构域和VP启动子的-288至-421结构域的进一步解剖，以确定每个基因中负责这些基因在SON中细胞特异性表达的<25bp的长序列。有了这些<25bp的DNA序列，应该可以使用酵母-1杂交技术来找出并鉴定与这些顺式序列结合的转录因子（tf）。

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