REGULATION OF THE CART GENE BY PROMOTER CIS-ELEMENTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 从病理学上讲，长期摄入滥用药物会导致"大脑奖励途径"的永久性遗传改变。“药物滥用研究的一个主要重点是确定该大脑区域受药物滥用影响的靶基因，并确定基因产物在成瘾表型中发挥的确切作用。 一个靶基因定位在大脑的奖励途径，并调节可卡因，是CART（可卡因和安非他明调节的转录本）基因，最初被确定为一个诱导型基因，其mRNA产物上调后，在大鼠延髓核（NAc）的精神兴奋剂管理。 我们实验室将解决的具体问题是：1）CART如何在大鼠NAc，垂体和培养细胞中转录调节，2）可卡因如何影响CART在大鼠NAc和垂体中的转录调节，3）CREB是否调节大鼠NAc中的CART？ 该项目的目的是--目的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、AP 1和Pit 1顺式调节元件结合; 2）可卡因狂欢治疗对CREB、Pit 1和AP 1 TF与上述顺式元件结合的影响（4 × 20 mg/kg，2小时）。目标二：使用野生型和突变型HSV-CREB构建体来确定大鼠NAc中CREB的过表达是否由于P-CREB与CART CRE顺式元件的结合增加而有助于CART肽水平的增加。目的3：通过染色质免疫沉淀法，在大鼠NAc中检测CREB和P-CREB是否与CART启动子CRE位点结合。到目前为止，目标1是完整的，我们的可卡因狂欢方案不影响CART基因转录调控。 目的2几乎完全，HSV-CREB确实引起CART肽的增加，并且该增加与通过Western印迹检测到的CREB蛋白水平的增加一致。

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.