Regulation of Oxytocin and Vasopressin Gene Expression in the hypothalamus.

我们正在解决两个关键问题和相关的假设有关的分子机制，调节细胞特异性OT和VP基因表达在下丘脑。 第一个问题是评估OT和VP基因中哪些顺式元件负责细胞特异性调控。先前的转基因研究（Young等，1990; Jeong等，2001; Davies等，2003年）和使用生物射弹和器官型培养的体外分析（Fields等，2003）表明，OT和VP的表达是由于OT基因中转录起始位点（TSS）上游554 bp处发现的特异性顺式元件的协调作用，VP基因TSS上游<3.4kbp5，VP和OT基因外显子3下游分别为178和430 bp。最近的体外研究表明，178和430 bp的下游调控元件（RE）在OT和VP基因的表达方面是可互换的（Fields，未发表），表明这些RE不负责细胞特异性表达。使用AAV载体在大鼠SON中用563-OT-III-EGFP-520序列体内转染（转染）神经元的更近期的实验已经证实，OT基因中TSS上游563 bp的DNA序列能够在OT大细胞神经元（MCN）中选择性地产生稳健的表达（Fields & Ponzio，未发表）。另外的实验已经表明，含有OT基因的448 bp、325 bp和216 bp上游序列的AAV载体可以支持细胞特异性OT基因表达（相对于VP细胞表达）。然而，216 bp的上游区域也导致SON背侧的非MCN神经元群体中的表达。相反，100 bp的上游区域产生清晰的表达，但完全没有特异性，这可能是核心启动子区域的预期。鉴于这些数据，我们假设，特定的顺式调控元件（RE）位于325 bp的OT基因的5上游区域，是至关重要的细胞特异性和调节表达的OT大细胞神经元（MCNs）在SON。更具体地说，我们假设：1）在OT基因的-216至-100区域中存在阻止VP细胞表达的阻遏物RE，2））在OT基因的-216至-100区域中存在对OT细胞表达特异的增强子RE，3）在OT基因的-325至-216区域中存在另一个阻遏物RE，其阻止在神经元的上SON（非MCN）群体中的表达，和4）在OT基因的-440至-216区域中可能存在对OT细胞表达特异的额外增强子RE。最后，先前在OT基因中描述的上游-180至-160bp处的推定ROR α激活位点（Chu和Zingg，1999）是OT基因的-216至-100区域中对OT细胞表达特异的推定增强子RE的候选者。正在进行的测试这些断言的实验是：1）确定SON中基底细胞特异性表达所必需的最小RE（325对216）是否也将支持急性和慢性渗透刺激期间的调节表达。 2)确定SON中基底细胞特异性表达所必需的最小RE（325对216）是否也将在PVN和SCN中起作用。 3)进行更多的实际和计算机模拟的缺失实验，以进一步剖析区域-325至-216和-216至-100中的推定RE。(It通过从p440 bp中去除-216至-100并观察没有-216至-100bp区域的所得序列是否在OT和VP细胞中都是稳健的，可以测试在-440至-216中是否存在额外的OT增强子和仅一个VP阻遏物，即在-216至100 bp中。5)开始VP基因中3.4kbp 5区域的缺失分析，以确定哪些RE对于SON中的细胞特异性和受调节的表达是关键的。 第二个问题是确定哪些转录因子和共调节因子存在于SON中的OT & VP MCNs中并在其中起作用。关于这个问题，我们先前使用了单细胞OT & VP表达文库的差异分析。（Yamashita等，2002）和SON和微阵列的激光显微切割。（Mutsuga等人，2004; 2005; Yue等人，2006），并且我们鉴定了除了OT和VP之外的许多特异性分子，其优先在SON中的MCN中表达，并且其在渗透适应过程中的表达基本上受到调节。我们假设，MCNs中OT和VP以及其他基因的细胞特异性表达将取决于每种细胞类型中转录体中的特异性蛋白质（例如，转录因子、增强子、抑制子、共激活子、共抑制子等）。解决该假设的正在进行的实验是：1）检查特定的候选转录因子、增强子、抑制子、共激活子、共抑制子基因是否实际上存在于SON中，以及它们是否选择性地在OT与VP MCN中表达。一种方法是对SON进行激光显微切割，分离其RNA，并通过qRTPCR确定SON中候选转录因子mRNA的存在。 作为SON中OT & VP转录的调节因子，我们将测试的一些候选转录因子是CREB、ER β、GR和ROR α。该策略将是使用慢病毒和/或AAV载体转导以在体内SON中表达野生型基因或敲低它们的表达（使用shRNA或显性负性特异性基因），然后通过qRTPCR测量OT & VP hnRNA的变化。

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.