Massie Charles E, Mills Ian G
Uro-Oncology Research Group, Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK.
Methods Mol Biol. 2009;505:123-37. doi: 10.1007/978-1-60327-575-0_7.
The identification of direct nuclear hormone receptor gene targets provides clues to their contribution to both development and cancer progression. Until recently, the identification of such direct target genes has relied on a combination of expression analysis and in silico searches for consensus binding motifs in gene promoters. Consensus binding motifs for transcription factors are often defined using in vitro DNA binding strategies. Such in vitro strategies fail to account for the many factors that contribute significantly to target selection by transcription factors in cells beyond the recognition of these short consensus DNA sequences. These factors include DNA methylation, chromatin structure, posttranslational modifications of transcription factors, and the cooperative recruitment of transcription factor complexes. Chromatin immunoprecipitation (ChIP) provides a means of isolating transcription factor complexes in the context of endogenous chromatin, allowing the identification of direct transcription factor targets. ChIP can be combined with site-specific PCR for candidate binding sites or alternatively with cloning, genomic microarrays or more recently direct high throughput sequencing to identify novel genomic targets. The application of ChIP-based approaches has redefined consensus binding motifs for transcription factors and provided important insights into transcription factor biology.
直接核激素受体基因靶点的鉴定为其在发育和癌症进展中的作用提供了线索。直到最近，此类直接靶基因的鉴定一直依赖于表达分析与在基因启动子中通过计算机搜索共有结合基序相结合的方法。转录因子的共有结合基序通常使用体外DNA结合策略来定义。此类体外策略未能考虑到许多对细胞中转录因子选择靶点有重大贡献的因素，而不仅仅是识别这些短的共有DNA序列。这些因素包括DNA甲基化、染色质结构、转录因子的翻译后修饰以及转录因子复合物的协同募集。染色质免疫沉淀（ChIP）提供了一种在内源染色质背景下分离转录因子复合物的方法，从而能够鉴定直接的转录因子靶点。ChIP可与针对候选结合位点的位点特异性PCR相结合，或者与克隆、基因组微阵列或最近的直接高通量测序相结合，以鉴定新的基因组靶点。基于ChIP的方法的应用重新定义了转录因子的共有结合基序，并为转录因子生物学提供了重要见解。
