Dam mutants provide improved sensitivity and spatial resolution for profiling transcription factor binding

DamID, in which a protein of interest is fused to Dam methylase, enables mapping of protein-DNA binding through readout of adenine methylation in genomic DNA. DamID offers a compelling alternative to chromatin immunoprecipitation sequencing (ChIP-Seq), particularly in cases where cell number or antibody availability are limiting. This comes at a cost, however, of high non-specific signal and a lowered spatial resolution of several kb, limiting its application to transcription factor-DNA binding. Here we show that mutations in Dam, when fused to the transcription factor Tcf7l2, greatly reduce non-specific methylation. Combined with a simplified DamID sequencing protocol, we find that these Dam mutants allow for accurate detection of transcription factor binding at a sensitivity and spatial resolution closely matching that seen in ChIP-seq. Overall design: Adenine methylation profiles following expression of Dam-Tcf7l2 or Dam only constructs with wild-type, N126A, or R95A variants of Dam in mouse embryonic stem cells. A negative (uninduced) control is included.

DamID技术是将目标蛋白与Dam甲基转移酶（Dam methylase）融合，通过检测基因组DNA中的腺嘌呤甲基化信号，实现蛋白-DNA结合位点的全基因组定位。相较于染色质免疫沉淀测序（ChIP-Seq），DamID是一种极具吸引力的替代技术，尤其适用于细胞数量受限或抗体可获得性不足的研究场景。但该技术也存在明显短板：非特异性信号较强，且空间分辨率仅为数kb，这极大限制了其在转录因子-DNA结合研究中的应用。本研究发现，当Dam发生突变并与转录因子Tcf7l2融合时，可大幅降低非特异性甲基化水平。结合简化的DamID测序实验流程，此类Dam突变体能够精准检测转录因子结合位点，其灵敏度与空间分辨率可与ChIP-Seq高度匹配。
总体实验设计：在小鼠胚胎干细胞中，分别表达携带野生型、N126A或R95A Dam变体的Dam-Tcf7l2融合蛋白，以及仅表达Dam的构建体，随后检测其腺嘌呤甲基化谱；同时设置阴性（未诱导）对照。