The NuRD Chromatin-Remodeling Enzyme CHD4 Promotes Embryonic Vascular Integrity by Transcriptionally Regulating Extracellular Matrix Proteolysis
Figure 3
CHD4 differentially regulates Plaur and Thbs1 expression in endothelial cells.
(A) qPCR with gene-specific primers for Plau, Plaur, and Thbs1 was performed on endothelial cells isolated from E10.5 littermate control and Chd4fl/fl;Tie2-Cre+ embryos. Data were normalized to the relative expression of control samples. Error bars represent SD of results from three independent experiments. (B and C) C166 endothelial cells were transfected with nonspecific (NS) or CHD4-specific siRNA oligonucleotides for 24 h. (B) Western blot analysis was performed on cell lysates using antibodies that recognize CHD4 or GAPDH. A representative blot from 3 independent experiments is shown. (C) RNA was isolated, cDNA was synthesized, and qPCR was performed using Plau-, Plaur-, or Thbs1-specific primers. Data were normalized to the relative expression of NS siRNA-treated samples. Error bars represent SD of results from three to four independent experiments. (D) Chromatin immunoprecipitation (ChIP) assays were carried out in C166 endothelial cells using antibodies against normal mouse IgG (negative control), CHD4, or HDAC1. Immunoprecipitated DNA was analyzed by qPCR to examine CHD4 and HDAC1 enrichment at the Plau, Plaur, and Thbs1 promoters. A transcriptionally inactive region approximately 5 kb upstream of the Fzd5 transcription start site (Fzd5UP) was assessed as a negative control for CHD4 and HDAC1 binding. Data are represented as a percent of total input chromatin. Error bars represent SD of results from three independent experiments. Results for Plau and Fzd5UP ChIP experiments are magnified in the insets. For the Plaur and Thbs1 ChIPs, CHD4 and HDAC1 binding were statistically compared against IgG binding at the respective loci or against CHD4 and HDAC1 binding at the Fzd5UP locus; both sets of comparisons revealed significant enrichment. (E) qPCR with gene-specific primers for Chd4, Plaur, and Thbs1 was performed on C166 endothelial cells transfected with 0.02 ng of a CHD4 expression plasmid or the analogous empty vector backbone (control). Data were normalized to the relative expression of control samples. Error bars represent SD of results from three independent experiments. (F) Schematic of the region of the murine Plaur promoter that was cloned into a luciferase (LUC) reporter plasmid for use in G. The Plaur promoter fragment encompasses the region to which CHD4 and HDAC1 were shown to bind by ChIP in D. (G) Luciferase assays were performed in C166 cells co-transfected with 250 ng of the reporter schematized in F and 10 ng of a constitutive Renilla luciferase control plasmid. Cells were also transfected with either 10 pmol of non-specific (NS) siRNA or CHD4 siRNA oligonucleotides to knock down endogenous CHD4 or with the CHD4 expression plasmid or its relevant control (empty vector) described in E. All transfections were performed for 24 h. Ratios of relative luciferaseā¶renilla activity were normalized to results from the control samples. Error bars represent SD of results from four independent experiments (with triplicate samples) for the siRNA-transfected samples and from five independent experiments (with triplicate samples) for the CHD4/control plasmid-transfected samples. All statistical calculations for Figure 3 were performed using a two-tailed Student's t test (*, p<0.05).