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

免疫球蛋白(Ig)对于维持对多种病原体的免疫力是必不可少的。 然而，关于化学物质或疾病状态对免疫球蛋白基因表达的影响，人们知之甚少。我们 研究表明，2，3，7，8-四氯二苯并-对二恶英(TCDD)是一种著名的B细胞抑制因子 分化，有效地抑制下游发现的转录调节区的激活 IgH基因座(3‘LGH RR)。除了被认为在免疫球蛋白基因表达中的作用外，3‘LGH RR也被 与特定的人类病理有关，包括Burkitt淋巴瘤、IgA肾病和腹腔积液 疾病。TCDD和相关化学品的许多毒性效应都归因于 芳香烃受体(AhR)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。我们检测到AhR与 3‘LGH RR的两个增强剂hs1、2和hs4内的矿点，发现这些矿点密切相关 与核因子-ICB结合基序相关。我们假设TCDD抑制3‘LGH-RR的激活 通过与Kb基序结合的NF-ISB/Rel蛋白复合体中的AhR依赖的位移 Hs1、hs2和hs4增强子。以下具体目标(SA)将检验这一假设。SA#1： 确定3‘lgHH RR中的哪个元件(S)需要激活和TCDD诱导 利用受3‘LGH RR和Cre-loxP技术调控的IGH微基因座进行抑制。SA#2： 确定TCDD是否通过抑制AhR来抑制3‘LGH RR的激活 以AhR基因为靶点的siRNA表达。SA#3：确定TCDD和内毒素诱导的结合 EMSA-Western和CHIP分析hs4和hs1，2增强子中的核因子-KB/Rel蛋白。 SA#4：确定核因子-KB/Rel蛋白是否介导TCDD对3‘LGH RR激活的抑制作用 通过用KBA超级阻遏蛋白抑制这些蛋白或通过过度表达特定的核因子- Kb/Rel融合蛋白。SA#5：确定人类hs1，2增强子的多态是否对TCDD敏感 人类hs1，2增强子调控的荧光素酶报告基因诱导抑制。建议数 研究将为我们阐明生理和病理的长期目标提供基础。 AhR和NF-KB/Rel蛋白在人3‘LGH调节中的作用(由AhR配体诱导) RR及其增强剂及其与人类疾病的关系。这些研究的结果也将是 适用于其他AhR激动剂(和拮抗剂)的危害评估，包括广泛的 来自环境、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.