Figure 3: H2A.X removal from HCT116 cells leads to enhanced transcriptional activity of Slug and ZEB1.

(a) H2A.X depletion enhanced the promoter activity of Slug and ZEB1, but not GAPDH. Slug, ZEB1 and GAPDH promoter activities were accessed by luciferase reporter assay in control cells (shCTRL), in cells silenced for H2A.X (shH2A.X), in parental cells (H2A.X WT) and in H2A.X knockout cells (H2A.X KO). Error bars represent the s.e.m. (n=3). Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (b) H2A.X binding to Slug and ZEB1 promoter using chromatin immunoprecipitation (ChIP) assay. Chromatin from control cells (shCTRL) and cells silenced for H2A.X (shH2A.X) or parental cells (H2A.X WT) and H2A.X knockout cells (H2A.X KO) was immunoprecipitated with anti-H2A.X antibody. The purified DNA was analysed by real-time PCR using primers amplifying across Slug, ZEB1 and GAPDH promoters. Results are presented as percentage of total input DNA precipitated. GAPDH promoter serves as an internal control. Error bars represent the s.e.m. (n=3). Statistical significance was determined by a two-tailed, unpaired Student’s t-test (c) H2A.X deletion enhances the enrichment of H3K9ac to Slug and ZEB1 promoters. Cells used in b were processed for ChIP using anti-H3K9ac antibody. GAPDH promoter serves as an internal control. Error bars represent the s.e.m. (n=3). Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (d) Hypothetical model for the role of H2A.X in the transcriptional regulation of Slug and ZEB1 during EMT in colon cancer cells. H2A.X removal from the nucleosome leads to enhanced enrichment of active chromatin marks (H3K9ac) within the promoters of Slug and ZEB1. This chromatin configuration enables the transcriptional activation of Slug and ZEB1. Elevated levels of Slug and ZEB1 are key in mediating the expression of several EMT-related genes.