ARID1a (BAF250), a component of human SWI/SNF chromatin remodeling complexes, is frequently mutated across numerous cancers, and its loss of function has been putatively linked to glucocorticoid resistance. Here, we interrogate the impact of siRNA knockdown of ARID1a compared to a functional interference approach in the HeLa human cervical cancer cell line. We report that ARID1a knockdown resulted in a significant global decrease in chromatin accessibility in ATAC-Seq analysis, as well as affecting a subset of genome-wide GR binding sites determined by analyzing GR ChIP-Seq data. Interestingly, the specific effects on gene expression were limited to a relatively small subset of glucocorticoid-regulated genes, notably those involved in cell cycle regulation and DNA repair. The vast majority of glucocorticoid-regulated genes were largely unaffected by ARID1a knockdown or functional interference, consistent with a more specific role for ARID1a in glucocorticoid function than previously speculated. Using liquid chromatography-mass spectrometry, we have identified a chromatin-associated protein complex comprising GR, ARID1a, and several DNA damage repair proteins including P53 binding protein 1 (P53BP1), Poly(ADP-Ribose) Polymerase 1 (PARP1), DNA damage-binding protein 1 (DDB1), DNA mismatch repair protein MSH6 and splicing factor proline and glutamine-rich protein (SFPQ), as well as the histone acetyltransferase KAT7, an epigenetic regulator of steroid-dependent transcription, DNA damage repair and cell cycle regulation. Not only was this protein complex ablated with both ARID1a knockdown and functional interference, but spontaneously arising DNA damage was also found to accumulate in a manner consistent with impaired DNA damage repair mechanisms. Recovery from dexamethasone-dependent cell cycle arrest was also significantly impaired. Taken together, our data demonstrate that although glucocorticoids can still promote cell cycle arrest in the absence of ARID1a, the purpose of this arrest to allow time for DNA damage repair is hindered.

ARID1a (BAF250) 是人类 SWI/SNF 染色质重塑复合物的一个组成部分，在多种癌症中经常发生突变，其功能丧失被认为与糖皮质激素耐药有关。在这里，我们探讨了 siRNA 敲低 ARID1a 与功能性干扰方法在 HeLa 人宫颈癌细胞系中的影响。我们报告说，ARID1a 敲低导致 ATAC-Seq 分析中染色质可及性整体显着下降，并影响通过分析 GR ChIP-Seq 数据确定的全基因组 GR 结合位点的子集。有趣的是，对基因表达的具体影响仅限于糖皮质激素调节基因的相对较小的子集，特别是那些涉及细胞周期调节和 DNA 修复的基因。绝大多数糖皮质激素调节基因基本上不受 ARID1a 敲低或功能干扰的影响，这与 ARID1a 在糖皮质激素功能中比之前推测的更具体的作用一致。使用液相色谱-质谱法，我们鉴定了一种染色质相关蛋白复合物，包含 GR、ARID1a 和几种 DNA 损伤修复蛋白，包括 P53 结合蛋白 1 (P53BP1)、聚 (ADP-核糖) 聚合酶 1 (PARP1)、DNA 损伤-结合蛋白 1 (DDB1)、DNA 错配修复蛋白 MSH6 和剪接因子脯氨酸和富含谷氨酰胺的蛋白 (SFPQ)，以及组蛋白乙酰转移酶 KAT7（类固醇依赖性转录、DNA 损伤修复和细胞周期调节的表观遗传调节因子） 。不仅这种蛋白质复合物因 ARID1a 敲低和功能干扰而被消除，而且还发现自发产生的 DNA 损伤以与受损的 DNA 损伤修复机制一致的方式积累。 从地塞米松依赖性细胞周期停滞中恢复也受到显着损害。综上所述，我们的数据表明，尽管糖皮质激素在缺乏 ARID1a 的情况下仍然可以促进细胞周期停滞，但这种停滞为 DNA 损伤修复留出时间的目的受到阻碍。