ChIP-chip analysis of GBF1 in the presence and absence of its heterodimerizing partners, HY5 and HYH. Arabidopsis thaliana

Transcription factors are key components of light signalling as they amplify the signal, which results in massive changes in genome-wide expression during photomorphogenesis. Three bZIP transcription factors (TFs), namely GBF1, HY5, and HYH, form heterodimers with each other and regulate photomorphogenesis in an interdependent manner. GBF1 acts as both a positive and negative regulator of photomorphogenesis, whereas HY5 and HYH mainly act as positive regulators of photomorphogenesis. In this study, the impact of heterodimerization of GBF1 with HY5 and HYH was analyzed for genome-wide binding of GBF1 through ChIP-chip in GBF1. We identified more than 2000 direct targets of GBF1 in the presence of HY5 and HYH. However, in the absence of HY5, very few binding sites were found, and in the absence of functional HYH protein, the number of GBF1 direct targets reduced to only half compared to when functional HYH was present. Overall design: ChIPed DNA with GBF1 antibody from GBF1OE, hy5 GBF1OE, and hyh GBF1OE lines vs. ChIPed DNA with GBF1 antibody from wild-type.

转录因子是光信号传导通路的核心组分，它们能够介导信号放大，进而在光形态建成（photomorphogenesis）过程中引发全基因组基因表达的显著变化。3个碱性亮氨酸拉链（bZIP）转录因子（TFs），分别为GBF1、HY5与HYH，可彼此形成异二聚体，并以相互依赖的方式调控光形态建成。GBF1可同时作为光形态建成的正、负调控因子，而HY5与HYH则主要充当光形态建成的正调控因子。本研究通过染色质免疫沉淀芯片（ChIP-chip）技术，分析GBF1与HY5、HYH的异二聚化对GBF1全基因组结合特性的影响。在HY5与HYH共同存在的条件下，我们共鉴定出超过2000个GBF1的直接靶标位点；然而，在缺失HY5的样本中，仅能检测到极少的GBF1结合位点；而在功能性HYH蛋白缺失的情况下，GBF1的直接靶标数量相较于功能性HYH存在时仅剩余一半。实验整体设计：以野生型样本中使用GBF1抗体富集得到的染色质免疫沉淀DNA为对照，分别对比分析GBF1过表达（GBF1OE）株系、hy5突变体背景下的GBF1过表达株系（hy5 GBF1OE）以及hyh突变体背景下的GBF1过表达株系（hyh GBF1OE）中，使用GBF1抗体富集得到的染色质免疫沉淀DNA样本。