NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets. Mus musculus

NKX2-5 is a homeodomain transcription factor that plays a central role in the cardiac gene regulatory network, and is commonly mutated in human congenital heart disease. Here, we take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for both wild type NKX2-5 and a mutation lacking the homeodomain (NKX2-5delHD), the latter to model loss-of-function in gene regulatory network. NKX2-5delHD bound hundreds of targets including NKX2-5 wild type targets and a unique set of “off-targets”, and retained partial functionality. We showed that NKX2-5delHD could heterodimerize with NKX2-5 wild type and cofactors, including ubiquitous ETS family members ELK1 and ELK4, through a tyrosine-rich homophilic interaction domain (YRD). NKX2-5delHD off-targets, but not those of an NKX2-5 YRD mutant, were enriched in ETS motifs and were occupied by ELK1/ELK4 proteins, as determined by DamID. Our study reveals unexpected activities for NKX2-5 mutations on chromatin, guided by interactions with their normal cardiac and general cofactors, and suggest potential for a novel type of gain-of-function in congenital heart disease. The supplementary bed file contains all binding regions detected for the N/C-terminal fusions reported in the manuscript, in addition to probe locations, ready to upload directly into UCSC browser (mm9). Overall design: DamID-chip TF binding analysis. N and C-terminal Dam fusions to ELK1, ELK4 and SRF vs. Input (dam-only) in HL-1 atrial cardiomyocytes. N-terminal Dam fusions to NKX2-5, NKX2-5YRD^(Y-A) and NKX2-5ΔHD (homeodomain deletion) vs. Input (dam-only) in HL-1 atrial cardiomyocyte.

NKX2-5是一种同源框结构域（homeodomain）转录因子，在心脏基因调控网络中发挥核心作用，且在人类先天性心脏病中常出现致病突变。本研究采用功能基因组学方法探究先天性心脏病的发病机制。我们通过DamID技术为野生型NKX2-5以及缺失同源框结构域的突变体NKX2-5delHD构建了一套可靠的靶基因集合，后者用于模拟基因调控网络中的功能丧失型突变。NKX2-5delHD可结合数百个靶位点，其中既包含NKX2-5野生型的靶标，也包含一套独特的"脱靶"位点，且仍保留部分功能。我们证实，NKX2-5delHD可通过一个富含酪氨酸的同源相互作用结构域（tyrosine-rich homophilic interaction domain，简称YRD）与野生型NKX2-5以及辅因子（包括普遍存在的ETS家族成员ELK1和ELK4）发生异二聚化。通过DamID实验分析发现，NKX2-5delHD的脱靶位点（而非NKX2-5 YRD突变体的脱靶位点）富集有ETS基序，且可被ELK1/ELK4蛋白结合。本研究揭示了NKX2-5突变在染色质上的意外活性，该活性由其与正常心脏辅因子及通用辅因子的相互作用所介导，并提示先天性心脏病中存在一类新型功能获得型突变的潜在可能。
补充的bed文件包含了本文报道的N端、C端融合蛋白的所有检测到的结合区域，同时附带探针位置信息，可直接上传至UCSC基因组浏览器（mm9版本）。
整体实验设计为DamID-chip转录因子结合分析：在HL-1心房心肌细胞中，分别构建ELK1、ELK4及SRF的N端、C端Dam融合蛋白，并以仅表达Dam的细胞作为对照；同时在HL-1心房心肌细胞中构建NKX2-5、NKX2-5YRD^(Y-A)及NKX2-5ΔHD（同源框结构域缺失突变体）的N端Dam融合蛋白，同样以仅表达Dam的细胞作为对照。