Genome-wide screen of transcription co-regulators identifies GLIS2 as a repressor for p53 target genes [ChIP-seq]

GLIS2, a member of the sub-family of Krüppel-like zinc finger proteins, has been linked to the development of nephronophthisis, while few studies have been conducted on the connection between tumorigenesis and GLIS2. In the current study, we identified GLIS2 as a repressor for p53 target genes, such as PUMA. GLIS2 ChIP-seq demonstrated that GLIS2 binds directly on PUMA promoter. In addition, GLIS2 inhibits the binding of p53 on PUMA promoter region and the modification of H3K27ac on PUMA enhancer. Taken together, GLIS2 is identified as an oncogene in colorectal cancer. Overall design: ChIP-seq for p53, H3K27ac, and p300 was performed in HCT116 followed by treatment with 375µM 5-FU and 10µM Nutlin3a for 8 h. GLIS2 ChIP-seq was performed by Dynabeads MyOne streptavidin C1 (Thermo-Fisher 65001). HCT116 cells were transfected with two different siRNAs of GLIS2 followed by treatment with 5-FU, ChIP-seq for p53, H3K27ac, H3K4me1 and H3K4me3 was performed.

GLIS2属于Krüppel样锌指蛋白（Krüppel-like zinc finger proteins）亚家族成员，已被证实与肾消耗病（nephronophthisis）的发生发展密切相关，但目前针对GLIS2与肿瘤发生之间关联的研究仍较为匮乏。本研究证实，GLIS2可作为PUMA等p53靶基因的抑制因子。GLIS2染色质免疫共沉淀测序（ChIP-seq）结果显示，GLIS2可直接结合于PUMA启动子区域。此外，GLIS2能够抑制p53结合至PUMA启动子区域，同时阻断PUMA增强子区域的组蛋白H3赖氨酸27乙酰化（H3K27ac）修饰。综上，本研究确认GLIS2在结直肠癌中作为癌基因发挥作用。

整体实验设计：在经375μM 5-氟尿嘧啶（5-FU）与10μM Nutlin3a处理8小时的HCT116细胞中，开展了p53、H3K27ac及p300的染色质免疫共沉淀测序；GLIS2染色质免疫共沉淀测序采用Dynabeads MyOne链霉亲和素C1磁珠（赛默飞世尔科技，货号65001）完成。将两种靶向GLIS2的小干扰RNA（siRNA）转染至HCT116细胞，经5-FU处理后，开展p53、H3K27ac、组蛋白H3赖氨酸4单甲基化（H3K4me1）及组蛋白H3赖氨酸4三甲基化（H3K4me3）的染色质免疫共沉淀测序。