Nucleosome driven transcription factor binding and gene regulation [microarray]. Homo sapiens

Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions, identified as DNaseI-hypersensitive sites (DHS). Here we show by chromatin-IP, MNase and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins we identified 25,000 PR binding sites (PRbs), the majority encompassing several copies of the hexanucleotide TGTYCY, highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes mainly in enhancers, are enriched in DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation. Keywords: time course Overall design: Whole genome expression microarrays were performed in T47D-MTVL cells stimulated 0, 1 and 6 hr with 10 nM of progestin R5020. 3 biological replicas were performed for T0 and 6 hr treatments and 4 for the one of 1hr.

阐明转录因子的全局功能，需鉴定其靶基因与基因组结合位点。此过程中染色质的作用尚不明确，但主流观点认为转录因子优先结合被鉴定为脱氧核糖核酸酶I超敏位点（DNaseI-hypersensitive sites, DHS）的核小体缺失区域。本研究通过染色质免疫沉淀（chromatin-IP）、微球菌核酸酶（MNase）及脱氧核糖核酸酶I（DNaseI）实验结合深度测序，证实孕酮受体（progesterone receptor, PR）的最佳结合与功能发挥依赖核小体。在经孕激素处理的乳腺癌细胞中，我们共鉴定出25000个PR结合位点（PRbs），其中大多数包含多拷贝的六核苷酸基序TGTYCY——该基序在基因组中丰度极高。研究发现，功能性PR结合位点主要富集于孕激素诱导基因周边的增强子区域，虽富集于DHS区域，但同时呈现较高的核小体占据率。孕激素刺激可引发这些核小体重构，导致组蛋白H1及H2A/H2B二聚体发生解离。本研究结果有力表明，核小体对于PR结合与激素介导的基因调控至关重要。
关键词：时间序列
实验设计概况：以10 nM孕激素R5020分别刺激T47D-MTVL细胞0、1及6小时后，进行全基因组表达微阵列检测。T0与6小时处理组各设置3次生物学重复，1小时处理组设置4次生物学重复。