NEGATIVE REGULATORY ELEMENTS IN TRANSCRIPTION OF HUMAN EPSILON GLOBIN GENE

人类 EPSILON 珠蛋白基因转录中的负调控元件

• 批准号: 2572901
• 负责人: A N SCHECHTER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2572901
• 关键词: DNA footprinting; developmental genetics; gene deletion mutation; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; globin; hemoglobin; human genetic material tag; posttranscriptional RNA processing; transcription factor; transfection

The first of two developmental switches in human hemoglobin expression involves the down-regulation or silencing of the embryonic globin gene (e). We have previously reported on a silencer element in the 5' flanking region of the e gene located at -270 bp 5' of the e-globin gene which provides negative regulation for this autonomously regulated gene. We have now extended this analysis 5' up to the globin locus control region (LCR) using reporter gene constructs with deletion mutations and transient transfection assays. Deletion of HS1 had minimal effect on e-globin promoter activity. We identified two negative regulatory regions: eNRA at -3 kb which increases transcription activity by thirty fold in erythroid K562 cells when deleted, and eNRB at -1.7 kb which appears to be erythroid specific. eNRA contains 55% evolutionarily conserved sequences and can reduce transcription activity of a minimal e-globin promoter or the SV40 promoter by two fold or more. Sequence and DNase I footprinting analyses indicate that this region contains a GATA-1 binding motif. Cotransfection of GATA-1 with an e-globin/luciferase construct containing eNRA leads to increased transcription activity, suggesting that the positive effect of GATA-1 dominates over any negative effect associated with the GATA-1 binding motif in the several negative control elements located in this 5' flanking region. For eNRB, DNA binding analyses indicate that a TATA-like binding motif is located within this region. We suggest that the down-regulation of e-globin gene expression as development progresses involves cooperative interaction of the negative regulatory elements, eNRA, eNRB, the e- globin silencer and possibly other negative and positive elements in the 5' flanking region of the e-globin gene.

人类血红蛋白表达的两个发育转变中的第一个 涉及胚胎珠蛋白基因的下调或沉默 (e). 我们之前曾报道过 5' 中的消音器元件 e 基因的侧翼区域位于 e 珠蛋白的 -270 bp 5' 对此自主提供负调控的基因 调控基因。 我们现在已将此分析扩展到 5' 至 使用报告基因构建体的球蛋白位点控制区（LCR） 缺失突变和瞬时转染测定。 删除 HS1 对e-珠蛋白启动子活性影响极小。 我们确定了两个 负调控区域：-3 kb 处的 eNRA 增加 红系 K562 细胞中的转录活性增加了 30 倍 已删除，-1.7 kb 处的 eNRB 似乎是红细胞特异性的。 eNRA 包含 55% 的进化保守序列，可以减少 最小 e-珠蛋白启动子或 SV40 的转录活性 启动子的两倍或更多。 序列和 DNase I 足迹 分析表明该区域包含 GATA-1 结合基序。 GATA-1 与 e-珠蛋白/荧光素酶构建体的共转染 含有 eNRA 会导致转录活性增加，表明 GATA-1 的积极作用胜过任何消极作用 与 GATA-1 结合基序相关的几个负 控制元件位于该 5' 侧翼区域。对于 eNRB、DNA 结合分析表明 TATA 样结合基序位于 在这个区域内。 我们建议下调e-globin 随着发育的进展，基因表达涉及合作 负调控元件 eNRA、eNRB、e- 的相互作用 珠蛋白消音器和可能的其他负面和正面元素 e-珠蛋白基因的 5' 侧翼区域。