REGULATION OF THE ID GENES IN B CELL DEVELOPMENT

B 细胞发育中 ID 基因的调控

• 批准号: 2390364
• 负责人: Xiao-Hong Sun
• 金额: $ 14.29万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390364
• 关键词: B lymphocyte; DNA footprinting; animal genetic material tag; antisense nucleic acid; cell cycle proteins; cell differentiation; flow cytometry; gel mobility shift assay; gene mutation; gene rearrangement; genetic regulation; genetic regulatory element; genetically modified animals; immunoglobulin genes; laboratory mouse; neoplastic transformation; tissue /cell culture; transcription factor

The differentiation of eukaryotic cells requires a hierarchy of regulatory mechanisms that involve a cascade of alteration in the transcription of specific genes. For example, specific gene expression (e.g., immunoglobulin genes) in pre-B and mature B cells is activated by a family of bHLH (basic helix-loop-helix) transcription factors. In progenitor B cells, however, the function of these bHLH transcription factors is suppressed by a group of inhibitory Id proteins whose expression diminishes only when the progenitor B cells are triggered to differentiate. The primary objective of my proposal is to understand the hierarchy of transcriptional events that dictates B cell development, through studying the regulation of the Id genes and the consequences of their dysregulation during B cell development. The findings of these studies may also shed light on mechanisms of differentiation operating in other cell types. Because the Id proteins are thought to be inhibitors of cell differentiation, knowledge about the regulation of the Id genes in B cell development may help to understand certain developmental aberrations that result in immunodeficiency, autoimmunity and malignancy. This research proposal includes the following two specific aims: (1) Investigation of the molecular mechanism of transcriptional regulation of the Id1 gene, including the identification of cis-acting elements and trans-acting factors for the down-regulation of the Id1 gene during B cell differentiation. The flanking sequences of the Id1 gene will be analyzed by means of deletion and point mutations and assayed for enhancer and silencer activities in B cell lines. The proteins binding to the identified cis-acting elements will be characterized by in vitro footprinting, in vivo genomic footprinting and electrophoretic mobility shift assays. (2) Determination of the effects of altered Id gene expression on the transcription of B cell-specific genes and the rearrangement of the immunoglobulin genes. The Id cDNAs will be over- expressed in pre-B and mature B cells using various expression vectors and Id expression will be reduced in pro-B cells by antisense oligonucleotides or antisense RNA. We will also generate transgenic mice which carry Id genes driven by constitutive B cell-specific promoters such as that of the mb-1 gene. We will determine where dysregulated Id expression in these transgenic mice can block B cell differentiation by monitoring the development of mature B cells using FACS analyses of bone marrow and spleen, and whether such blockage of differentiation causes certain malignancy in the immune system using hematopathological analyses of blood and bone marrow.

真核细胞的分化需要分级的调节 涉及转录级联改变的机制 特定基因。 例如，特定的基因表达（例如， 前 B 细胞和成熟 B 细胞中的免疫球蛋白基因）由家族激活 bHLH（碱性螺旋-环-螺旋）转录因子。 在祖细胞B中 然而，这些 bHLH 转录因子的功能是 被一组抑制性 Id 蛋白抑制，其表达 仅当祖 B 细胞被触发时才会减少 区分。 我的建议的主要目标是了解 决定 B 细胞发育的转录事件层次结构， 通过研究 Id 基因的调控及其后果 B 细胞发育过程中的失调。 这些研究结果 研究还可能揭示分化的机制 其他细胞类型。 因为 Id 蛋白被认为是 细胞分化，B 中 Id 基因调控的知识 细胞发育可能有助于理解某些发育异常 导致免疫缺陷、自身免疫和恶性肿瘤。 本研究计划包括以下两个具体目标：（1） 转录调控分子机制研究 Id1 基因，包括顺式作用元件的鉴定和 B 细胞过程中 Id1 基因下调的反式作用因子 差异化。 将分析 Id1 基因的侧翼序列 通过缺失和点突变并测定增强子和 B 细胞系中的沉默子活性。 结合到的蛋白质 鉴定出的顺式作用元件将通过体外表征 足迹、体内基因组足迹和电泳迁移率 转变分析。 (2)改变Id基因的影响的测定 B细胞特异性基因转录的表达及 免疫球蛋白基因的重排。 Id cDNA 将过度 使用各种表达载体在前 B 细胞和成熟 B 细胞中表达 反义寡核苷酸会降低 pro-B 细胞中的 Id 表达 或反义RNA。 我们还将培育携带 Id 的转基因小鼠 由组成型 B 细胞特异性启动子驱动的基因，例如 mb-1 基因。 我们将确定这些中 Id 表达失调的位置 转基因小鼠可以通过监测 B 细胞分化来阻断 使用 FACS 分析骨髓和成熟 B 细胞的发育 脾脏，以及这种分化受阻是否会导致某些 使用血液的血液病理学分析确定免疫系统中的恶性肿瘤 和骨髓。