Fig 1: Foxp3 binds to the Il9 locus and inhibits IL-9 expression by recruiting histone deacetylases.(a) Graph depicting Il9 locus and putative Foxp3 binding sites (red) at the Il9 promoter and CNS regions. (b) ChIP analysis of Foxp3 binding to the Il9 gene locus in CD4+ T cells transduced with retrovirus expressing GFP alone (Ctrl) or GFP-Flag-Foxp3 (Foxp3), then cultured under iTreg-polarizing conditions in the presence of DTA-1. GFP+ cells were sorted and fixed for ChIP using anti-Flag antibody. Data represent mean values±s.d. (n=3). (c,d) Flow cytometry plots showing IL-9-producing cells in CD4+ T cells transduced with retrovirus expressing GFP alone or Foxp3-GFP, and cultured under iTreg-polarizing conditions for 3 days in the presence of DTA-1. Numbers indicate the percentage of cells in the quadrants (c). Graph depicts the percentage of IL-9+ cells in GFP+ population (d). Data represent mean values±s.d. (n=6). (e) Co-immunoprecipitation of flag-foxp3 in CD4+ T cells transduced with retrovirus expressing flag-foxp3, and cultured under iTreg-polarizing conditions for 2 days. Anti-flag and control IgG immunoprecipitates (IP) were subjected to immunoblot analysis (IB) with anti-HDAC1, anti-HDAC2, anti-Sirt1 and anti-Sirt7 antibodies, and representative plots of 3 experiments are shown. (f) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in GFP+ cells sorted from naive CD4+ T cells transduced and activated as in (b) for 2 days. Data represent mean values±s.d. (n=3). (g,h) Flow cytometry analysis of IL-9-producing cells in naive CD4+ T cells transduced and activated as in (c) for 3 days in the presence or absence of HDAC inhibitor Trichostatin A (TSA, 2 nM) and Sirt1 inhibitor EX-527 (EX, 0.5 μM). Numbers in the quadrants indicate the percentage of positive cells (g). Graph depicts the percentage of IL-9+ cells in GFP+ population. Data represent mean values±s.d. (n=9) (h). (i) ChIP analysis of H3Ac and H4Ac modifications at Il9 promoter and CNS regions in Foxp3−GFP+ cells sorted from naive CD4+ T cells transduced with retrovirus expressing Foxp3-GFP and cultured as in (g) for 2 days. Data represent mean values±s.d. (n=3). P values were determined by Student's t-test (*P<0.05).
Fig 2: HDAC2 regulates oxidative stress to participate in proliferation and apoptosis(A) HDAC2 knockdown repressed high glucose-induced ROS accumulation in HUVECs. Transduced HUVECs with/without HDAC2 knockdown were treated with high glucose (HG, 30 mM) for 12 h, then the cells were subjected to analysis of total cellular ROS level. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (B) HDAC2 overexpression promoted high glucose-induced ROS accumulation in HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) for 12 h, then the cells were subjected to analysis of total cellular ROS level. *P<0.05, **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (C) NAC reduced the HDAC2-mediated increase in ROS level in high glucose-treated HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) and/or NAC (1 mM) for 12 h, then the cells were subjected to analysis of total cellular ROS level. *P<0.05, **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (D) MnTBAP reduced the HDAC2-mediated increase in ROS level in high glucose-treated HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) and/or MnTBAP (100 μM) for 12 h, then the cells were subjected to analysis of total cellular ROS level. *P<0.05, **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (E) NAC reduced the HDAC2-mediated increase in apoptosis in high glucose-treated HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) and/or NAC (1 mM) for 48 h, then the cells were subjected to analysis of apoptosis. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (F) MnTBAP reduced the HDAC2-mediated increase in apoptosis in high glucose-treated HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) and/or MnTBAP (100 μM) for 48 h, then the cells were subjected to analysis of apoptosis. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (G) NAC and MnTBAP blocked the effects of HDAC2 on apoptotic signaling pathways in high-glucose-treated HUVECs. Transduced HUVECs with/without HDAC2 overexpression were treated with high glucose (HG, 30 mM) and/or NAC (1 mM) or MnTBAP (100 μM) for 24 h, then the cells were subjected to Western blot analysis. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests.
Fig 3: HDAC2 regulates the expression of MnSOD in ECs(A) Knockdown of HDAC2 increases the expression of MnSOD. HUVECs were infected with a retrovirus carrying shRNA targetting HDAC2 for 48 h, then the mRNA and protein levels were analyzed. **P<0.01 by one-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (B) Overexpression of HDAC2 reduces the expression of MnSOD. HUVECs were infected with a retrovirus carrying HDAC2 for 48 h, then the mRNA and protein levels were analyzed. **P<0.01 by unpaired Student’s t test. (C) Representative ChIP results showing HDAC2 binds the promoter of MnSOD. HUVECs were subjected to ChIP assay with anti-IgG or anti-HDAC2 antibodies, followed by qRT-PCR experiments to detect the binding of HDAC2 to MnSOD promoter. **P<0.01 by unpaired Student’s t test. (D) High glucose promotes the binding of HDAC2 to MnSOD promoter. HUVECs were treated with/without high glucose (HG, 30 mM) for 24 h and the cells were subjected to ChIP assay with anti-IgG or anti-HDAC2 antibodies, followed by qRT-PCR experiment to detect the enrichment of HDAC2 at MnSOD promoter. **P<0.01 by unpaired Student’s t test. (E) High glucose reduces H3K9Ac and H3K27Ac at MnSOD promoter. HUVECs were treated with/without high glucose (HG, 30 mM) for 24 h and the cells were subjected to ChIP assay with anti-H3K9Ac or anti-H3K27Ac antibodies, followed by qRT-PCR experiment to detect the enrichment of H3K9Ac and H3K27Ac at MnSOD promoter. **P<0.01 by unpaired Student’s t test. (F) Representative Western blot showing MnSOD knockdown in HUVECs. HUVECs were infected with a retrovirus carrying shRNA targetting MnSOD for 48 h. (G) MnSOD knockdown blocks the effects of HDAC2 knockdown on the high glucose-induced accumulation of ROS in HUVECs. Transduced HUVECs with/without HDAC2 and/or MnSOD knockdown were treated with high glucose (HG, 30 mM) for 12 h, then the cells were subjected to analysis of total cellular ROS level. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests. (H) MnSOD knockdown blocks the effects of HDAC2 knockdown on high glucose-induced apoptosis in HUVECs. Transduced HUVECs with/without HDAC2 and/or MnSOD knockdown were treated with high glucose (HG, 30 mM) for 48 h, then the cells were subjected to analysis of apoptosis. **P<0.01 by two-way ANOVA followed by Bonferroni post hoc multiple comparisons tests.
Fig 4: Rescue by Hdac2 KO of gene expression levels of a subset of genes dysregulated by the HttQ111 allele.(A) To identify genes dysregulated by the HttQ111 allele and by Hdac2 KO in HttQ111/+ mice, the overlapping genes in contrasts 3 and 2 were identified (left table). This shows the numbers of genes (p<0.05) up-regulated by HttQ111 and either further up-regulated, or down-regulated by Hdac2 KO in HttQ111/+ mice, and the numbers of genes down-regulated by HttQ111 and either further down-regulated, or up-regulated by Hdac2 KO in HttQ111/+ mice. To identify HttQ111-dysregulated genes whose expression level was normalized by Hdac2 KO, genes from the contrast 3/2 overlap whose expression level did not differ significantly (p>0.05) between Htt+/+ Hdac2 WT and HttQ111/+ Hdac2 KO striata were identified (right table). (B) Heat map of the 97 genes down-regulated by HttQ111 and up-regulated by Hdac2 KO in HttQ111/+ striata and of the 55 genes up-regulated by HttQ111 and down-regulated by Hdac2 KO in HttQ111/+ striata, whose expression levels did not differ significantly between Htt+/+ Hdac2 WT and HttQ111/+ Hdac2 KO. (C) Examples of four such genes, two down-regulated by HttQ111 and two up-regulated by HttQ111 are displayed as box-plots.
Fig 5: RUNX1 and RUNX1/ETO Complexes Differentially Interact with Coactivator and Corepressor Complexes, and Binding to the Same Sites Is Mutually Exclusive(A–E) Multiple RUNX1/ETO binding sequences and control sequences (IVL, Chr18) were selected and validated for factor binding by a first round of ChIP followed by a second round with a different antibody or with just beads as indicated. All of the chosen binding sites contain several RUNX1 motifs (data not shown).(A) LMO2 associates with both RUNX1 and RUNX1/ETO.(B and C) RUNX1 and RUNX1/ETO binding is mutually exclusive. Control ChIPs were performed with the same antibody.(D) EP300 associates with RUNX1, but not RUNX1/ETO.(E and F) RUNX1 preferentially binds p300, whereas RUNX1/ETO preferentially associates with HDAC2. For additional amplicons, see Figure S2B. qPCR data represent the mean ± SD of at least three independent experiments.
Supplier Page from Abcam for Anti-HDAC2 antibody