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

说明(申请人提供)：免疫球蛋白(Ig)对于保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。我们已经证明，2，3，7，8-四氯二苯并-对二恶英(TCDD)是一种众所周知的B细胞分化抑制因子，它能有效地抑制IgH基因下游转录调节区(3‘LGH RR)的激活。除了在免疫球蛋白基因表达中的作用外，3‘LGH RR还与特定的人类病理有关，包括Burkitt淋巴瘤、IgA肾病和腹腔疾病。TCDD和相关化学物质的许多毒性效应归因于芳烃受体(AhR)的激活导致基因表达的变化，该受体随后与受影响基因中的二恶英反应元件(DRE)结合。我们在3‘LGH RR、hs1、2和HS4的2个增强子中检测到AhR与DRE位点的结合，并发现这些DRE位点与NF-kB结合基序密切相关。我们假设TCDD通过依赖AhR改变与hs1、2和HS4增强子中的kB基序结合的NF-kB/Rel蛋白复合体来抑制3‘LGH RR的激活。以下具体目标(SA)将检验这一假设。SA#1：利用由3‘lGH RR和Cre-loxP技术调控的IgH微基因座，确定3’lgHH RR中哪些元件(S)需要激活和TCDD诱导的抑制。SA#2：确定TCDD通过靶向AhR基因的siRNA抑制AhR表达来抑制3‘LGH RR激活是否依赖于AhR。SA#3：通过EMSA-Western和CHIP分析，确定TCDD和LPS诱导的NF-kB结合谱假设TCDD通过依赖于AhR的HS4和HS1，2增强子中的NF-kB/Rel蛋白的位移抑制3‘LGH RR的激活。SA#4：确定是通过用IkBA超阻遏蛋白抑制这些蛋白还是通过过度表达特定的NF-kB/REL融合蛋白来介导TCDD对3‘LGH RR激活的抑制作用。SA#5：用人类hs1，2增强子调控的荧光素酶报告基因确定多态的人hs1，2增强子是否对TCDD诱导的抑制敏感。这些研究将为我们的长期目标奠定基础，即阐明AhR和NF-1B/Rel蛋白在调节人类3‘LGH RR及其增强子中的生理和病理作用及其与人类疾病的关系。这些研究的结果也将适用于其他AhR激动剂(和拮抗剂)的危险评估，包括来自环境、饮食和药物来源的广泛化学物质。

DESCRIPTION (provided by applicant): 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 (DRE) in the affected genes. We have detected binding of AhR to DRE sites within 2 enhancers of the 3'lgH RR, hs1,2 and hs4, and find that these DRE sites are closely associated with NF-kB binding motifs. We hypothesize that TCDD represses 3'lgH RR activation through an AhR-dependent shift in the NF-kB/Rel protein complexes binding to kB motifs within the hs1,2 and 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 hypothesize that TCDD represses 3'lgH RR activation through an AhR-dependent shift in the 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 IkBa 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-?B/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.