Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer

We report the chromatin binding sites of HOXB7 transcription factor in BT-474 breast cancer cell line using ChIP-sequencing. We validated the chromatin binding sites in BT-474, MDA-MB-361, MCF7 and T-47D breast cancer cell lines using ChIP-qPCR. The ChIP experiments have been performed using HOXB7 antibody and IgG non-specific antibody as a negative control. The direct downstream target genes of HOXB7 were identified by analyzing the expression of genes located nearby HOXB7 binding sites in HOXB7 knockdown versus control cells using qRT-PCR. Overall design: Examination of chromatin binding sites of HOXB7 in BT-474 breast cancer cell line using ChIP-seq. Four parallel IgG samples were sequenced, merged together and used as a control data set. Two parallel HOXB7 ChIP samples were sequenced and merged for each replicate, AF1 and AF2. Both HOXB7 ChIP replicates (AF1 and AF2) contained approximately the same amount of reads as the merged IgG control data set.

本研究报道了BT-474乳腺癌细胞系中同源盒B7（HOXB7）转录因子的染色质结合位点，相关检测采用染色质免疫共沉淀测序（ChIP-sequencing，简称ChIP-seq）技术。本研究通过染色质免疫共沉淀-实时定量PCR（ChIP-qPCR），对BT-474、MDA-MB-361、MCF7及T-47D这4种乳腺癌细胞系中的染色质结合位点进行了验证。本次染色质免疫共沉淀实验以HOXB7抗体作为靶标抗体，并以IgG非特异性抗体作为阴性对照。为鉴定HOXB7的直接下游靶基因，本研究通过实时定量反转录PCR（qRT-PCR），分析了HOXB7敲低组与对照组细胞中HOXB7结合位点邻近基因的表达水平。

总体实验设计：采用ChIP-seq检测BT-474乳腺癌细胞系中HOXB7的染色质结合位点。对4份平行IgG对照样本进行测序，将测序数据合并后作为对照数据集。针对两个生物学重复（AF1与AF2），分别对每个重复内的2份平行HOXB7 ChIP样本进行测序，并将同重复内的平行样本测序数据合并。两个HOXB7 ChIP生物学重复（AF1与AF2）的测序读段（reads）总数量，与合并后的IgG对照数据集的测序读段总数量大致相当。