Figure 3.

ChIP assay of NF-Y binding to the Dmrt7 promoter in testis. a), Western blot analysis of the Dmrt7 protein in the adult testis, heart, liver, spleen, lung, kidney, ovary, brain and epididymis. The Dmrt7 expression was only detected in the testis. β-actin was used as an internal control. b), Interaction of NF-Y with the Dmrt7 promoter in vivo was determined by chromatin immunoprecipitation analysis. Samples of mouse testis and liver were chopped into small pieces and cross-linked in 1% formaldehyde to cross-link endogenous proteins and DNA. Samples of sonicated chromatin were immunoprecipitated with anti-NFYA, no antibody (beads only) and preimmuno IgG (control) respectively. DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 160-bp sequence of mouse Dmrt7 promoter corresponding to the -101 to +59 region. Primers for an unrelated region were used as negative control. The amplified PCR fragments were analyzed on 2% agarose gel. c), A q-ChIP PCR was performed to quantify the binding extent of NF-Ya on the Dmrt7 promoter. All values were initially expressed relative to relevant IgG DNA content. d), The relative positions of the primers are shown.