Regulation of Oxytocin and Vasopressin Gene Expression in the hypothalamus.

病毒载体方法通常被用作体内将基因转移到神经系统的手段，并被非常有效地用于将基因转移到下丘脑神经元和MCNs中。此外，病毒载体已被成功地用于研究参与体内细胞特异性基因表达的基因启动子区域。我们在我们的器官型下丘脑培养模型中测试了几种类型的病毒载体的转导效率，发现腺相关病毒(AAV)在体外能够非常有效地转导OTMCNs，而没有任何毒性证据。因此，我们选择使用包含OT和AVP基因启动子缺失结构的AAV载体，融合到EGFP报告中在体内转导大鼠子体内的MCNs。 我们使用包含OT或VP基因启动子缺失结构的AAV载体与EGFP报道融合在一起，在体内转导(转染)大鼠SON中的神经元。将这些动静脉动静脉注射到大鼠子脑中，两周后进行EGFP的表达，然后灌流固定大鼠的脑，最后对下丘脑的冷冻切片进行免疫组织化学染色，以评估EGFP在OT和VP-MCNs中的表达情况。 OT基因的研究：注射含有568-OT-III-EGFP-520序列的AAVs表明，OT基因转录起始点(TSS)上游的568bp DNA序列能够选择性地在OT-MCNs中表达，但不能在VP-MCNs中表达。进一步的实验表明，含有OT基因启动子上游448bp、325bp和216bp序列的AAV载体都能支持OT-MCNs中细胞特异性的OT基因表达(但不支持VPMCNs中的表达)。50bp和100bp的上游区域在SON中确实产生了EGFP的表达，但在OT-和VP-MCNs中弱而非选择性地表达，这可能是一个核心启动子区域。此外，我们还发现OT基因中的内含子1和2以及外显子2和3不是细胞特异性表达所必需的。这些数据最近发表在菲尔兹等人的2012年《公共科学图书馆·综合》上。 VP基因的研究：我们扩展了AAV缺失研究，以检测VP启动子中的顺式结构域。我们构建了一个含有2kb启动子的VP载体，该启动子通过外显子1直接连接到EGFP报告基因，并发现立体定向注射该AAV仅在VP-MCNs中产生了强劲的EGFP表达。随后在VP启动子中的缺失表明在1到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288bp以下。在OT和VP缺失研究中有一个令人惊讶的发现，这些基因的渗透调节机制在所有的缺失结构中都存在，这表明这种调节存在于核心启动子区域，可能位于POL II结合部位。针对VP启动子-288bp区域下方进一步的缺失实验表明，负责表达特异性的RE位于VP启动子的-288~-100bp区域内。这些研究已经完成，刚刚提交发表(Ponzio等人，提交给PLoS one)。 从上述研究中我们得出结论，这里描述的AAV方法是一种在实验上研究中枢神经系统细胞类型特异性基因表达的高效方法，并且可以很容易地应用于任何感兴趣的基因和大脑区域。 随着负责基因细胞类型特异性基因表达的调控元件开始被鉴定，未来的研究将需要确定与这些元件结合的转录因子(即转录因子结合位点)。显然，需要对OT和VP启动子进行进一步的缺失实验，以便更好地定义通常构成TFBS的特定8-10个碱基序列。我们开始了一项关于SON中OT和VP MCNs中存在哪些转录因子和协同调控因子的研究，利用我们通过上述AAV策略选择性地在OT-和VP-MCNs中表达EGFP的能力，作为解决这一问题的新机会。我们分别注射rAAV-p440-OT-EGFP和rAAV-p2kb-VP-EGFP选择性地标记OT-MCNs或rAAV-p2kb-VP-EGFP选择性标记Vp-MCNs，并用激光显微切割(LCM)分离SON中的荧光MCNs进行分子分析。然后，我们从每种细胞类型的单细胞解剖池中分离出RNA，并通过qRTPCR确定OT和VP MCNs中候选转录因子mRNAs的相对数量。我们对RORA、CREB3、ARNT1、CLOCK和AP1等5个候选转录因子进行了研究，发现OT-MCNs的RORA是VPMCNs的两倍。这是一个重要的原则证明，特别是因为假设的RORA激活位点位于先前报道的-156bp的上游，因为OT基因是推测的增强子/抑制物RE的首选候选，预测位于OT基因的-216到-100区域特异性的OT-MCN表达(Humerick等人正在准备一篇描述这些实验的论文)。 最近，我们使用LCM/RNA-SEQ方法进行了实验，以比较通过我们对OT和VP基因中已识别的相关顺式结构域中TF结合位点的生物信息学分析所预测的假定TF的存在，以及从激光显微解剖的SON中提取的RNA获得的RNA-SEQ数据(手稿正在准备中，Johnson等人和Shii等人)。 该项目将在下一财年终止。

Viral vector methods have been routinely used as a means to deliver genes into the nervous system in vivo, and have been very effectively used for gene transfer into hypothalamic neurons and in MCNs. In addition, viral vectors have been successfully used to study gene promoter domains that are involved in cell-specific gene expression in vivo. We tested the transduction efficiencies of several types of viral vectors in our organotypic hypothalamic culture model and found that adeno-associated viruses (AAV), were able to transduce OT MCNs in vitro very effectively without any evidence of toxicity. Consequently, we choose to use AAV vectors that contain promoter deletion constructs of the OT and AVP gene promoters fused to EGFP reporters to transduce MCNs in the rat SON in vivo. We used 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. Studies on the OT gene: 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 upstream sequences of the OT gene promoter all can support cell-specific OT gene expression in OT-MCNs (but not in VP MCNs). The 50bp and 100bp upstream regions do produce EGFP expression in the SON but weakly and non-selectively in the OT-and VP- MCNs, which 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. These data were recently published in Fields, et al, PLoS One, 2012. Studies on the VP gene: We extended the AAV-deletion studies to examine the cis-domains in the VP promoter. 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 below 288bp. A 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 indicates that this regulation resides in the core promoter domain, possibly at the Pol II binding site. Deletion experiments directed at further dissections below the -288bp domain in the VP promoter indicated that the RE responsible for specificity of expression lies within the -288 to -100bp domain in the VP promoter. These studies have been completed and have just been submitted for publication (Ponzio et al, submitted to PLoS One. From the above studies we conclude that the AAV approach described here is a highly effective way to experimentally study cell-type specific gene expression in the central nervous system, and could easily be applied to any gene and brain region of interest. As the regulatory elements that are responsible for the cell-type specific gene expression of the genes begin to be identified, then future studies will need to determine the transcription factors (TFs) that bind to these elements (i.e, to the transcription factor binding sites, TFBSs). Clearly further deletion experiments are needed on the OT and VP promoters in order to better define the specific 8-10 base sequences that usually constitute TFBSs. We began a study of which transcription factors and co-regulators are present & functioning in the OT & VP MCNs in the SON, by using our ability to selectively express EGFP in the OT- and VP-MCNs by the AAV strategy described above, as a novel opportunity to approach this issue. 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 for five candidate TFs, e.g, RORA, CREB3, ARNT1 CLOCK, and AP1, and found 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 occurs at -156bp upstream as previously reported for the OT gene is a prime candidate for the putative enhancer/repressor RE predicted to be in the -216 to -100 region of the OT gene specific for OT-MCN expression (a paper describing these experiments is now in preparation, Humerick et al). More recently we have done experiments using an LCM/ RNA-seq approach to compare the presence of putative TFs predicted by our bioinformatic analyses of TF binding sites in the identified, relevant cis-domains in the OT and VP genes to the RNA-seq data obtained from RNA extracted from laser microdissected SONs (manuscripts in preparation, Johnson et al, and Shi et al). This project will terminate in the next fiscal year.