He Aibin, Pu William T
Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA.
Curr Protoc Mol Biol. 2010 Oct;Chapter 21:Unit 21.20. doi: 10.1002/0471142727.mb2120s92.
Recent development of methods for genome-wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. ChIP requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions. This unit provides a detailed protocol for genome-wide location analysis of in vivo biotinylated transcription factors by streptavidin pull-down followed by high-throughput sequencing (bioChIP-seq).
近期，通过染色质免疫沉淀法（ChIP）进行全基因组转录因子结合位点鉴定的方法取得了进展，这为转录调控带来了新的见解，并增进了对单个转录因子功能的理解。ChIP需要针对目标转录调节因子的高度特异性抗体，而合适抗体的可得性是该方法更广泛应用的重大障碍。通过用一种短生物表位标记目标转录调节因子，可以规避这一限制，该表位可被大肠杆菌BirA酶特异性生物素化。然后，在严格条件下，生物素化的转录因子可在链霉亲和素磁珠上被选择性地拉下。本单元提供了一个详细方案，用于通过链霉亲和素拉下法，随后进行高通量测序（bioChIP-seq），对体内生物素化的转录因子进行全基因组定位分析。
