National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2010 Dec 29;5(12):e15898. doi: 10.1371/journal.pone.0015898.
Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites.
使用基于染色质免疫沉淀（ChIP）的技术在全基因组范围内鉴定转录因子结合位点，在解决发育问题方面正成为一种越来越重要的工具。然而，在许多生物系统中，与因子丰度和合适的 ChIP 试剂相关的技术问题是这些研究的常见障碍。我们使用了两种完全不同的、广泛适用的方法，通过 ChIP 确定了线虫胚胎中主肌调节转录因子 HLH-1（CeMyoD）的全基因组结合位点。这两种方法，ChIP-seq 和 ChIP-chip，产生了强烈重叠的结果，表明 HLH-1 优先结合富含 E-box 序列（CANNTG）的基因的启动子区域，这是该研究充分的转录因子的已知结合位点。HLH-1 结合位点在上皮细胞中已知表达的基因上游富集，与它作为直接转录调节剂的作用一致。HLH-1 结合也在许多与肌肉基因表达无关的位点被检测到，这与其小鼠同源物 MyoD 先前的描述一致。这些结合位点可能反映了 HLH-1 的几个额外功能，包括与一个或多个辅助因子相互作用以激活（或抑制）基因表达，或者在直接转录调节靶基因之外发挥在染色质组织中的作用。我们的结果还提供了 ChIP 方法的比较，可以克服这些类型研究中常见的限制，同时突出将体内功能分配给鉴定的靶位点的复杂性。
