Chen Jiguo
Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA.
Methods Mol Biol. 2009;567:113-32. doi: 10.1007/978-1-60327-414-2_8.
The ability to determine genome-wide location of transcription factor binding sites (TFBS) is crucial for elucidating gene regulatory networks in human cells during normal development and disease such as tumorigenesis. To achieve this goal, we developed a method called serial analysis of binding elements for transcription factors (SABE) for globally identifying TFBS in human or other mammalian genomes. In this method, a specific antibody targeting a DNA-binding transcription factor of interest is used to pull down the transcription factor and its bound DNA elements through chromatin immunoprecipitation (ChIP). ChIP DNA fragments are further enriched by subtractive hybridization against non-enriched DNA and analyzed through generation of sequence tags similar to serial analysis of gene expression (SAGE). The SABE method circumvents the need for microarrays and is able to identify immunoprecipitated loci in an unbiased manner. The combination of ChIP, subtractive hybridization, and SAGE-type methods is advantageous over other similar strategies to reduce the level of intrinsic noise sequences that is typically present in ChIP samples from human or other mammalian cells.
确定转录因子结合位点（TFBS）在全基因组范围内的位置，对于阐明人类细胞在正常发育和诸如肿瘤发生等疾病过程中的基因调控网络至关重要。为实现这一目标，我们开发了一种名为转录因子结合元件序列分析（SABE）的方法，用于在人类或其他哺乳动物基因组中全局鉴定TFBS。在该方法中，使用针对感兴趣的DNA结合转录因子的特异性抗体，通过染色质免疫沉淀（ChIP）拉下转录因子及其结合的DNA元件。ChIP DNA片段通过与未富集的DNA进行消减杂交进一步富集，并通过生成类似于基因表达序列分析（SAGE）的序列标签进行分析。SABE方法无需微阵列，能够以无偏倚的方式鉴定免疫沉淀的位点。ChIP、消减杂交和SAGE类方法的结合比其他类似策略更具优势，可降低人类或其他哺乳动物细胞ChIP样本中通常存在的内在噪声序列水平。
