ETV6/RUNX1 inhibits MIR181A1 transcription via binding to the endogenous RUNX1 site.

(A) Schematic representation of the genomic structure of human MIR181A1. The location of the MIR181A1 gene and the RUNX1-binding site are numbered relative to the TSS (+1). Arrows indicate the locations of the primers used in the ChIP assay. (B) ChIP was carried out using anti-RUNX1 or in the absence of specific antibody (Control) (left). DNA sequences surrounding the putative RUNX1-binding site were amplified by PCR using P1 primers. To evaluate the specificity of RUNX1 binding, a positive control and a negative control were performed using PC and P2, respectively, for the ChIP assay. Amplification of the upstream region near the RUNX1-binding site on MIR223, which is a known direct target of RUNX1, was performed using PC primers. P2 primers were designed to amplify a distal region lacking the RUNX1-binding site. Input shows the amplification from sonicated chromatin, and genomic DNA (gDNA) was used as a positive PCR control. The PCR products were quantified by densitometry (right). (C) The ChIP assay was performed using anti-HDAC3 (left). Treatment with valproic acid and amplification of the promoter region of GAPDH were used as controls. The PCR products were quantified by densitometry (right). Bars show the mean ± SD from three independent experiments. *P ≤ 0.05, **P ≤ 0.01 (ANOVA).(A) 人类MIR181A1基因的基因组结构示意图。以转录起始位点（Transcription Start Site, TSS）的+1位点为参照基准，对MIR181A1基因的位置及RUNX1结合位点进行编号，箭头标示出本次染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验中所用引物的结合位点。(B) 本次实验分别采用抗RUNX1抗体以及不加特异性抗体的空白对照（Control）进行ChIP实验（左图）。使用P1引物对推定的RUNX1结合位点周边的DNA序列进行聚合酶链式反应（Polymerase Chain Reaction, PCR）扩增；为验证RUNX1结合的特异性，本次ChIP实验分别以PC引物作为阳性对照、P2引物作为阴性对照。使用PC引物对已知的RUNX1直接靶基因MIR223上RUNX1结合位点附近的上游区域进行扩增，P2引物则被设计用于扩增不含RUNX1结合位点的远端区域。Input组为超声破碎后的染色质模板的扩增产物，基因组DNA（gDNA）作为PCR阳性对照，通过光密度扫描法对PCR产物进行定量分析（右图）。(C) 采用抗组蛋白去乙酰化酶3（HDAC3）抗体完成本次ChIP实验（左图），以丙戊酸处理组以及甘油醛-3-磷酸脱氢酶（GAPDH）启动子区域的扩增产物作为对照，同样通过光密度扫描法对PCR产物进行定量分析（右图）。柱状图展示了三次独立实验的平均值±标准差（SD），其中*表示P ≤ 0.05，**表示P ≤ 0.01（方差分析（Analysis of Variance, ANOVA））。