3'alpha enhancer regulation by AhR and NF-kappaB/Rel proteins

免疫球蛋白（IG）对于维持针对多种病原体的免疫是必需的。 然而，关于化学物质或疾病状态对IG基因表达的影响知之甚少。我们 已经表明，2，3，7，8-四氯二苯并-p-二恶英（TCDD），一种众所周知的B细胞抑制剂， 分化，有效地抑制转录调控区下游的激活， IgH基因座（3 'IgH RR）。除了其在IG基因表达中的作用外，3 'lgH RR也被认为是一种特异性的蛋白质。 与特定人类病理学相关，包括伯基特淋巴瘤、伊加肾病和乳糜泻 疾病TCDD和相关化学品的许多毒性作用都归因于 由芳香烃受体（AhR）的激活引起的基因表达， 与受影响基因中的二恶英反应元件（ORE）结合。我们已经检测到AhR与 在3 'lgH RR的两个增强子hs 1，2和hs 4内的ORE位点，并发现这些ORE位点与3' lgH RR的两个增强子hs 1，2和hs 4紧密相关。 与NF-κ B结合基序相关。我们假设TCDD抑制3 'lgH RR激活， 通过NF-κ B/Rel蛋白复合物与KB基序结合的AhR依赖性转变， HS 1、HS 2和HS 4增强子。以下具体目标（SA）将检验这一假设。SA #1： 确定3 'lgHH RR中的哪些元素是激活和TCDD诱导的 利用由3 'lgH RR和CRE-loxP技术调节的IgH迷你基因座进行阻遏。SA #2： 通过抑制AhR来确定TCDD对3 'lgH RR激活的抑制是否依赖于AhR 用靶向AhR基因的siRNA表达。SA #3：测定TCDD和LPS诱导的结合 通过EMSA-Western和ChIP分析的hs 4和hs 1，2增强子内的NF-κ B/Rel蛋白的谱。 SA#4：确定NF-κ B/Rel蛋白是否介导TCDD对3 'lgH RR激活的抑制作用 通过用kBa超阻遏蛋白阻遏这些蛋白或通过特异性NF-κ B的过表达， KB/Rel融合蛋白。SA #5：确定多态性人hs 1，2增强子是否对TCDD敏感- 用受人HS 1，2增强子调节的荧光素酶报告基因诱导抑制。拟议 研究将为我们阐明生理和病理的长期目标提供基础。 （由AhR配体诱导）AhR和NF-κ B/Rel蛋白在人3 'IgH调节中的作用 RR及其增强子与人类疾病的关系。这些研究的结果也将 适用于其他AhR激动剂（和拮抗剂）的危害评价，包括广泛的 环境、饮食和药物来源的化学品。

Immunoglobulins (Ig)are essential for maintaining immunity against a wide variety of pathogens. However, little is known regarding the impact of chemicals or disease states on Ig gene expression. We have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known suppressor of B cell differentiation, potently inhibits activation of the transcriptional regulatory region found downstream of the IgH locus (3'lgH RR). In addition to its proposed role in Ig gene expression, the 3'lgH RR has also been associated with specific human pathologies including Burkitt's lymphoma, IgA nephropathy and Celiac disease. Many of the toxic effects of TCDD and related chemicals have been attributed to changes in gene expression resulting from the activation of the aryl hydrocarbon receptor (AhR) which subsequently binds to dioxin-responsive elements (ORE) in the affected genes. We have detected binding of AhR to ORE sites within two enhancers of the 3'lgH RR, hs1,2 and hs4, and find that these ORE sites are closely associated with NF-icB binding motifs. We hypothesize that TCDD represses 3'lgH RR activation through an AhR-dependent shift in the NF-isB/Rel protein complexes binding to KB motifs within the hs1,2and hs4 enhancers. The following specific aims (SA) will test this hypothesis. SA#1: Determine which element(s) within the 3'lgHH RR are required for activation and for TCDD-induced repression utilizing an IgH mini-locus regulated by the 3'lgH RR and CRE-loxP technology. SA#2: Determine if TCDD's inhibition of 3'lgH RR activation is dependent on the AhR by inhibiting AhR expression with siRNA targeted to the AhR gene. SA#3: Determine the TCDD and LPS-induced binding profile of NF-KB/Rel proteins within the hs4 and hs1,2 enhancers by EMSA-Western and ChIP analyses. SA#4: Determine whether NF-KB/Rel proteins mediate TCDD's repressive effect on 3'lgH RR activation by repression of these proteins with an kBa super represser protein or by over-expression of specific NF- KB/Rel fusion proteins. SA#5: Determine if the polymorphic human hs1,2 enhancer is sensitive to TCDD- induced inhibition with luciferase reporter genes regulated by the human hs1,2 enhancer. The proposed studies will provide the foundation for our long-term goal of elucidating the physiological and pathological (induced by AhR ligands) roles of the AhR and NF-KB/Rel proteins in the regulation of the human 3'lgH RR and its enhancers and their relation to human disease. Results of these studies will also be applicable to the hazard evaluation of other AhR agonists (and antagonists) which include a wide array of chemicals from environmental, dietary and pharmaceutical origin.