Figure 5: Histone acetylation/deacetylation of HBR regulates the expression of Nodal.

(a) ChIP–qPCR assay in day 2 cells. Enrichments of HDAC1 at the binding region (HBR) of the Nodal locus were normalized with the control region (∼1 kb downstream of HBR region). (b) Analysis of H3ac levels on the HBR in day 4 cells differentiated from WT or HDAC1 KO mESCs by H3ac ChIP–qPCR. (c) Analysis of H3ac levels on the HBR in day 3 cells treated with DMSO or TSA by H3ac ChIP–qPCR. (d) Analysis of H3ac levels on the HBR during mESC neural differentiation. (e) Analysis of H3K9ac levels on the HBR during mESC neural differentiation. (f) Analysis of phosphorylated RNA polymerase II (pPol II) binding on the promoter of Nodal during mESC neural differentiation. (g) Expression analysis of Nodal during mESC neural differentiation with DMSO or TSA treatment. Relative expression level was normalized by gapdh. (h) Statistical analysis of Sox1–GFP⁺ cells at day 4. Cells were differentiated from control WT 46C or HBR-deleted 46C (#15 and #34). (i) Expression analysis of Nodal during neural differentiation of control WT 46C or HBR-deleted 46C (#15 and #34). Relative expression level was normalized by gapdh. Representative data of ChIP–qPCR (a–f) and qPCR (g,i) were shown from three independent experiments. Data represent mean±s.d. *P<0.05, **P<0.01, Student’s t-test.