ChIP-Seq analysis to identify direct binding of ZNF165

We found that the cancer testis antigen, ZNF165, is required for viability and modulates TGFβ-induced gene expression in mesenchymal, Claudin-Low, TNBC. To begin to define ZNF165's role in TNBC tumorigenesis, we performed ChIP-Seq analysis in the mesenchymal TNBC tumor derived cell line, WHIM12, stably expressing ZNF165-V5. This analysis uncovered 381 peaks associated with 410 genes and included TGFβ target genes, SMURF2 and SMAD, that promote negative TGFβ feedback regulation. Our results provide insight into how ZNF165 globally modulates TGFβ signaling and nominates ZNF165 candidate gene targets. WHIM12 cells were stably infected ZNF165-V5 were grown to 75% confluency. Chromatin was isolated, sonicated and immunoprecipitated using a V5 antibody. Purifed ChIP-ed DNA was then sequence, aligned to hg19 and HOMER was justed to identify significantly enriched peaks.

我们发现，癌睾丸抗原锌指蛋白165（ZNF165）是细胞存活所必需的，且可在间叶型、紧密连接蛋白低表达型三阴性乳腺癌（TNBC）中调控转化生长因子β（TGFβ）诱导的基因表达。为明确ZNF165在三阴性乳腺癌发生发展中的作用，我们在稳定表达ZNF165-V5的间叶型三阴性乳腺癌来源细胞系WHIM12中开展了染色质免疫共沉淀测序（ChIP-Seq）分析。本次分析共鉴定出381个结合峰，关联410个编码基因，其中包含可促进转化生长因子β负反馈调控的TGFβ靶基因SMURF2与SMAD。本研究结果揭示了ZNF165全局性调控转化生长因子β信号通路的分子机制，并筛选出ZNF165潜在候选靶基因。将稳定转染ZNF165-V5的WHIM12细胞培养至汇合度75%，收集细胞并分离染色质，经超声破碎后使用V5抗体进行免疫沉淀。纯化所得的染色质免疫共沉淀DNA随后进行测序，将测序序列比对至人类参考基因组hg19，并使用HOMER工具鉴定出显著富集的结合峰。