Identify cis-regulatory modules for OCT4. Homo sapiens

Recent ChIP-chip studies have revealed that many in vivo binding sites have a weak match to the consensus sequence for the transcription factor being analyzed. Possible explanations for these observations include a) the consensus site was derived from in vitro analyses and does not represent the preferred in vivo binding site and/or b) the factor is recruited to a weak binding site via interaction with a protein that binds nearby. To address these possibilities we have developed a motif discovery approach (ChIPMotifs) that incorporates a bootstrap re-sampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from in vivo ChIP-chip data by ab initio motif discovery programs. Using OCT4 ChIP-chip data derived from genomic tiling arrays and the ChIPMotifs approach, we developed a refined OCT4 PWM. We then used the in vivo-derived PWM and a ChIPModules approach to identify transcription factors co-localizing with OCT4 in a testicular germ cell tumor (Ntera2 cells). We found that the consensus binding site for SRY, a transcription factor critical for testis development, co-localizes with the OCT4 PWM. To further characterize the relationship between OCT4 and SRY binding sites, we used ChIP-chip analysis of human promoter microarrays, and found that 49% of the top ~1000 OCT4 target promoters were also bound by SRY. This analysis represents the first identification of SRY target promoters. Our studies not only validate the ChIPMotifs and ChIPModules combinatorial approach but also identify a possible new regulatory partner of OCT4. Keywords: ChIP-chip, computational approaches Overall design: First performing ChIP-chip experiments, then find OCT4 binding motif, then identify regulatory modules using ChIPModules approach.

近期的染色质免疫共沉淀芯片（ChIP-chip）研究显示，诸多体内结合位点与所分析转录因子的共有序列仅存在较弱的匹配度。针对该现象的可能解释包括：a) 共有结合位点源自体外分析，并非体内偏好的结合位点；以及/或b) 该转录因子通过与邻近结合蛋白的相互作用，被招募至弱结合位点。
为解答上述疑问，本研究开发了一种基序发现（motif discovery）方法（ChIPMotifs），该方法整合了bootstrap重采样技术，可通过统计推断，为由从头基序发现程序（ab initio motif discovery programs）从体内ChIP-chip数据中鉴定得到的基序的位置权重矩阵（PWM）确定最优截断阈值。
利用源自基因组瓦片阵列（genomic tiling arrays）的OCT4的ChIP-chip数据与ChIPMotifs方法，我们构建了优化后的OCT4 PWM。
随后，我们借助该体内来源的PWM与ChIPModules方法，在睾丸生殖细胞肿瘤（Ntera2细胞）中鉴定出与OCT4共定位的转录因子。研究发现，调控睾丸发育的关键转录因子SRY的共有结合位点，与OCT4 PWM存在共定位现象。
为进一步解析OCT4与SRY结合位点之间的关联，我们利用人类启动子微阵列（promoter microarrays）开展了ChIP-chip分析，结果显示，排名前约1000的OCT4靶启动子中，有49%同时被SRY结合。本分析首次实现了SRY靶启动子的鉴定。
本研究不仅验证了ChIPMotifs与ChIPModules的联合分析策略，还发现了OCT4潜在的新型调控伴侣分子。
关键词：ChIP-chip，计算方法（computational approaches）
整体实验设计：首先开展染色质免疫共沉淀芯片实验，随后鉴定OCT4结合基序，最后通过ChIPModules方法识别调控模块（regulatory modules）。