EFECTS OF TRANSCRIPTION FACTORS ON EPSILON GLOBIN EXPRESSION

转录因子对 Epsilon Globin 表达的影响

• 批准号: 6161919
• 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/6161919
• 关键词: DNA binding protein; developmental genetics; embryonic stem cell; erythroid stem cell; gene expression; gene induction /repression; globin; human genetic material tag; site directed mutagenesis; transcription factor; transfection

The human epsilon-globin gene is transcribed in erythroid cells only during the embryonic stages of development. The developmental control of epsilon-globin gene is mediated by a transcriptional silencer characterized by cell transcription assays as the epsilon-globin gene silencer motif (epsilon GSM) located at -278 to -257 bp 5' to the cap site. Mutation in the GATA-1/YY1 sites within epsilon GSM resulted in expression of the human epsilon-globin gene in hAEC system as well as in transgenic mice. Recently, two other negative regulatory regions at - 3 kb (epsilon NRA) and at -1.7 kb (epsilon NRB) have been characterized in our laboratory; epsilon NRA contains two GATA motifs. To further understand the role of GATA transcription factors on epsilon-silencing, we have studied their effect on the epsilon-globin gene expression. We have transiently transfected embryonic erythroid K562 cells with increasing amounts of an expression vector for the human transcription factor GATA-1. We have observed that increasing amounts of GATA-1 transcripts have a negative effect on the level of expression of endogeneous epsilon-globin gene, while there is little or no effect in other globin genes, such as gamma or alpha globin or other genes containing functional GATA-binding sites in their promoter regions such as glycophorin C. We have also observed that the level of expression of transcription factor GATA-2 decreases as the expression of GATA-1 increases. By stable transfections, we have created K562 cell lines constitutively expressing high levels of transcription factors GATA-1 and confirmed the negative effect of increasing levels of GATA-1 on epsilon-globin gene expression. Interestingly, in K562 cells expressing high levels of GATA-2 transcription factor (through transient or stable transfections), increasing levels of endogenous epsilon-globin and gamma-globin genes are observed. RNAse protection assays are in progress to better characterize the effect of GATA-2 transcription factor on globin gene expression. These findings suggest that GATA-1 and -2 transcription factors might mediate transcriptional regulation of the epsilon-globin gene through a concentration/dependent mechanism. These should provide an interesting model to understand how quantitative changes in transcription factors may result in qualitative alteration in target gene expression.

人类ε珠蛋白基因仅在红系细胞中转录 在发育的萌芽阶段。发育控制 ε-珠蛋白基因的表达是由转录沉默子介导的 通过细胞转录测定表征为ε-珠蛋白基因 消音器基序 (epsilon GSM) 位于 -278 至 -257 bp 5' 处 地点。 epsilon GSM 内 GATA-1/YY1 位点的突变导致 人ε珠蛋白基因在hAEC系统中的表达以及 在转基因小鼠中。近期，另外两个负面监管区域 - 3 kb (epsilon NRA) 和 -1.7 kb (epsilon NRB) 已 在我们的实验室进行了表征； epsilon NRA 包含两个 GATA 基序。 进一步了解GATA转录因子在 epsilon-沉默，我们研究了它们对 epsilon-globin 的影响 基因表达。我们瞬时转染了胚胎红细胞 K562 细胞中含有越来越多的人类表达载体 转录因子GATA-1。我们观察到数量不断增加 GATA-1 转录本对表达水平有负面影响 内源性ε珠蛋白基因的影响，而影响很小或没有影响 在其他珠蛋白基因中，例如 γ 或 α 珠蛋白或其他基因 在其启动子区域含有功能性 GATA 结合位点，例如 作为血型糖蛋白C。我们还观察到表达水平 转录因子 GATA-2 随着 GATA-1 的表达而减少 增加。通过稳定转染，我们创建了K562细胞系 组成型表达高水平的转录因子 GATA-1 并证实了 GATA-1 水平增加的负面影响 ε-珠蛋白基因表达。有趣的是，在 K562 细胞中表达 高水平的 GATA-2 转录因子（通过瞬时或稳定 转染），增加内源性ε珠蛋白的水平和 观察到γ-珠蛋白基因。 RNAse 保护测定正在进行中 更好地表征 GATA-2 转录因子对 珠蛋白基因表达。 这些发现表明 GATA-1 和 -2 转录因子可能介导转录调控 ε-珠蛋白基因通过浓度/依赖性机制。 这些 应该提供一个有趣的模型来理解如何定量 转录因子的变化可能导致质的改变 在目标基因表达中。