INTERACTION OF CIS-ACTING ELEMENTS IN CD8 REGULATION

说明书（申请人的摘要）： CD 4-CD 8+（CD 8 SP）和肠上皮内淋巴细胞的分化 （IELs）仍然知之甚少。使用转基因技术的实验室的结果 一些方法已经描述了一种增强子（称为E81 驱动IEL和成熟CD 8 SP中报告基因表达的CD 8a基因 细胞，而不是在胸腺细胞在分化的早期阶段。体外 我们实验室的转染研究已经确定了大约200 nt 顺式作用元件（称为L2 a）位于CD 8a基因上游4-5 kb， 似乎是CD 8阳性杂交瘤中负调控的靶点。 与胸腺淋巴瘤细胞系BW 5 147融合的T细胞。L2 a元件是 核基质相关区（MAR）。有趣的是，包括两个 E81增强子和含有L2 a元件的4.3kb Hindlil/Hindlil片段 导致E8 I增强子在CD 4 + CD 8+中的功能 双阳性（DP）胸腺细胞以及CD 8 SP T细胞和IEL中。我们 假设是L2 a元件赋予E81增强子 在DP胸腺细胞中发挥作用的能力。为了验证这一假设，转基因 使用人CD 2报告基因和4.3 kb HindIII/HmdIII片段的研究 将使用带有各种缺失的DNA片段来定位赋予DP的区域， 胸腺细胞功能对E81增强子的影响。这些顺式作用的效果 序列对其他CD 8增强子活性的影响将使用 使用Cre/loxP系统的相同方法敲除和敲入研究将在 进行L2 a元件的缺失或修饰（或 其他顺式作用序列）在胸腺中表达CD 8后的原位表达 亚群和外周T细胞。如果L2 a元素被证明是 顺式作用序列作为假设，我们将专门修改网站内 L2 a与我们提出的MAR结合蛋白SATB 1和CDP/Cux结合 在CD 8a基因调控中分别起正性和负性作用。 SATB 1或CDP/Cux表达受损的补充研究 将试图确定观察到的效果是否与 当L2 a中每个蛋白质的结合位点被消除时可见。最后是染色质 将进行交联和免疫沉淀研究以测试是否 SATB 1和CDP/Cux与L2 a元件结合，细胞特异性一致 与我们假设的CD 8基因调控中的作用一致。这些 这些研究将有助于我们了解T细胞免疫过程中CD 8基因的调控。 分化，以及MAR及其同源结合所发挥的作用 蛋白质在体内调节顺式作用序列的活性。

DESCRIPTION (Applicant's Abstract): The regulation of CD8 expression during the differentiation of CD4-CD8+ (CD8SP) and intestinal intraepithelial lymphocytes (IELs) is still poorly understood. Results from laboratories using transgenic approaches have described an enhancer (called E81) approximately 16 kb upstream of the CD8a gene that drives reporter gene expression in IELs and mature CD8SP cells but not in thymocytes at earlier stages of differentiation. In vitro transfection studies in our laboratory have defined an approximately 200 nt cis-acting element (called L2a) located 4-5 kb upstream of the CD8a gene that appears to be the target of negative regulation in hybridomas of CD8-positive T-cells fused with the thymic lymphoma cell line, BW5 147. The L2a element is a nuclear matrix associated region (or MAR). Interestingly, inclusion of both the E81 enhancer and a 4.3 kb Hindlil/Hindlil fragment containing the L2a element in the transgenic construct results in E8I enhancer function in CD4+CD8+ double-positive (DP) thymocytes as well as in CD8SP T-cells and IELs. We hypothesize that it is the L2a element that imparts to the E81 enhancer the ability to function in DP thymocytes. To test this hypothesis, transgenic studies using a human CD2 reporter gene and 4.3 kb HindIII/HmdIII fragments with various deletions will be used to localize the region that imparts DP thymocyte function upon the E81 enhancer. The effect of these cis-acting sequences on the activity of other CD8 enhancers will be evaluated using the same approach knock-out and knock-in studies using the Cre/loxP system will be performed to test the effect of deletion or modification of L2a element (or other cis-acting sequences) in situ upon CD8 expression in thymic subpopulations and peripheral T-cells. If the L2a element proves to harbor the cis-acting sequences as hypothesized, we will specifically modify sites within L2a shown to bind the MAR-binding proteins, SATBI and CDP/Cux, suggested by us to play positive and negative roles, respectively, in CD8a gene regulation. Complementary studies in which expression of SATB1 or CDP/Cux is compromised will be attempted to determine whether the effect observed is similar to that seen when each protein's binding site in L2a is abolished. Finally, chromatin cross-linking and immunoprecipitation studies will be performed to test whether SATB 1 and CDP/Cux bind to the L2a element with cell specificity consistent with the roles we have hypothesized for them in CD8 gene regulation. These studies will contribute to our knowledge of CD8 gene regulation during T-cell differentiation, and to the roles played by MARs and their cognate binding proteins in modulating the activities of cis-acting sequences in vivo.