REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 从病理上讲，长期服用滥用药物会导致“大脑奖励途径”发生永久性的基因改变。药物滥用研究的一个主要焦点是确定该脑区受药物滥用影响的靶基因，并确定基因产物在成瘾表型中所起的确切作用。CART(可卡因和苯丙胺调节转录本)基因是一个定位于大脑奖赏通路并受可卡因调节的靶基因，最初被发现是一种诱导性基因，其mRNA产物在给予精神刺激药物后在大鼠伏隔核(NAC)中上调。我们实验室将解决的具体问题是：1)CART在大鼠NAC、垂体和培养细胞中是如何转录调节的；2)可卡因如何影响CART在大鼠NAC和垂体中的转录调节；以及3)CREB是否调节大鼠NAC中的CART？该项目的目标1：确定：1)来自大鼠NAC和垂体的转录因子(TF)是否与CART启动子Cre、AP1和Pit1顺式调控元件结合；以及2)暴食可卡因对CREB、Pit1和AP1 Tf与上述顺式元件结合的影响(4×20 mg/kg，2小时)。目的：利用野生型和突变型HSV-CREB结构来确定CREB在大鼠NAC中的过度表达是否导致CART多肽水平的增加，这是由于P-CREB与CART Cre顺式元件结合增加所致。目的：用染色质免疫沉淀法检测CREB和P-CREB在大鼠NAC体内是否与CART启动子Cre结合。到目前为止，目标1已经完成，我们的狂欢可卡因方案不会影响CART基因的转录调控。AIM 2几乎完成，HSV-CREB确实引起CART多肽的增加，并且这种增加与Western blotting检测到的CREB蛋白水平的增加是一致的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pathologically, the chronic intake of drugs of abuse causes permanent genetic alterations in the "brain reward pathway." A major focus of drug abuse research is to identify target genes affected by drugs of abuse in this brain region and to ascribe the precise role that gene product plays in the addicted phenotype. One target gene localized in the brain reward pathway, and regulated by cocaine, is the CART (cocaine- and amphetamine-regulated transcript) gene, originally identified as an inducible gene whose mRNA product is up regulated in the rat nucleus accumbens (NAc) after psychostimulant administration. The specific questions our laboratory will address are: 1) how is CART transcriptionally regulated in the rat NAc, pituitary and cultured cells, 2) how does cocaine affect the transcriptional regulation of CART in the rat NAc and pituitary, and 3) does CREB regulate CART in the rat NAc? The aims of the project are--Aim 1: to determine: 1) if transcription factors (TFs) from rat NAc and pituitary bind CART promoter CRE, AP1, and Pit1 cis-regulatory elements; and 2) the effect of binge cocaine treatment on CREB, Pit1 and AP1 TF binding to the above cis-elements (4x20mg/kg, 2hrs). Aim 2: to use wild-type and mutant HSV-CREB constructs to determine if over expression of CREB in the rat NAc contributes to increases in CART peptide levels due to increased P-CREB binding to the CART CRE cis-element. Aim 3: to determine if CREB and P-CREB bind to the CART promoter CRE site in vivo, in the rat NAc by chromatin immunoprecipitation. To date, aim 1 is complete and our binge cocaine regimen does not affect CART gene transcriptional regulation. Aim 2 is nearly complete and HSV-CREB does cause increases in CART peptide and that increase coincides with increased levels of CREB protein as detected by Western blotting.