Figure 1.
HDAC1 interacts with Fli1 and mediates its deacetylation. A.
A schematic representation of pCTAP-Fli1. Streptavidin and calmodulin resins bind to streptavidin-binding peptide (SBP) and calmodulin-binding peptide (CBP), respectively. ATA: A-terminal activation domain, EBD: Ets binding domain, CTA: C-terminal activation domain. B. Foreskin fibroblasts were grown to subconfluence and serum-starved for 48 hours. Equal amount of protein from whole cell lysates was subjected to immunoprecipitation with or without anti-Fli1 antibody. The levels of Fli1, HDAC1, and HDAC3 proteins in precipitates were determined by immunoblotting. The levels of Fli1, HDAC1 and HDAC3 in whole cell lysates were determined by Western blotting. C. 293 T cells were transfected with pCTAP expression vector encoding wild type Fli1 along with PCAF, PCAFΔHAT, or HDAC1 expression vectors, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of Flag-tagged proteins in cell lysates were determined by Western blotting. D. 293 T cells were transfected with pCTAP wild type Fli1 along with PCAF or PCAFΔHAT expression vectors. Six hours after the transfection, HDAC1 antisense oligonucleotide (HDAC1 AS oligo) or HDAC1 sense oligonucleotide (HDAC1 S oligo) were added to the cells. Whole cells lysates were prepared 48 hours after transfection. The levels of acetylated Fli1 and Flag-tagged proteins were determined as described above. The efficacy of HDAC1 AS oligo was evaluated by Western blotting.
Figure 2.
p300 increases the DNA binding ability of Fli1 through HDAC1-dependent deacetylation of lysine 380. A.
293T cells were transfected with pCTAP wild type Fli1 along with the indicated HAT proteins, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads), followed by immunoblotting using rabbit anti-acetylated lysine antibody (AcK). In order to visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of HAT proteins in cell lysates were determined by Western blotting. B. Whole cell lysates prepared as described above were incubated with biotin-labeled oligonucleotides. Proteins bound to these nucleotides were isolated with streptavidin-coupled agarose beads. The levels of Flag-tagged proteins and Fli1 in cell lysates were determined by Western blotting. C. 293T cells were transfected with pSG5 wild type Fli1 or Fli1 K380Q mutant along with the p300 expression vector, and incubated for 48 hours. D. 293T cells were transfected with pCTAP wild type Fli1 along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using SA beads, followed by immunoblotting using anti-HDAC1 antibody. To visualize the total levels of ectopically expressed Fli1, the same membrane was stripped and reprobed with anti-calmodulin binding peptide antibody. The levels of ectopically expressed p300 and endogenous HDAC1 in cell lysates were determined by Western blotting with anti-Flag antibody and anti-HDAC1 antibody, respectively.
Figure 3.
Phosphorylation of Fli1 at threonine 312 decreases its interactions with p300 and HDAC1. A.
293T cells were transfected with pCTAP wild type Fli1 or the Fli1 T312A mutant along with the p300 expression vector, and incubated for 48 hours. Total cell extracts were subjected to immunoprecipitation using streptavidin-coupled agarose beads (SA beads) followed by immunoblotting with anti-Flag antibody (left panels), or to immunoprecipitation using the anti-Flag antibody followed by immunoblotting with anti-calmodulin antibody (right panels). The levels of ectopically expressed p300 or Fli1 in cell lysates were determined by Western blotting with anti-Flag antibody (the bottom left panel) or anti-calmodulin antibody (the bottom right panel), respectively. B. Foreskin fibroblasts were transduced with 25 MOI of Fli1 or green fluorescence protein (GFP) adenovirus for 72 hours (left panels), or/and 25 MOI of Fli1siRNA or scrambled RNA (SCR) adenovirus for 72 hours (right panels). Nuclear extracts were subjected to immunoprecipitation with anti-p300 antibody, followed by immunoblotting with anti-HDAC1 antibody. The levels of Fli1, p300, and HDAC1 in nuclear extracts were evaluated by Western blotting. C. Whole cell lysates prepared from untreated or TGF-β stimulated foreskin fibroblasts were subjected to immunoprecipitation with anti-Fli1 antibody, followed by immunoblotting with antibodies against p300, HDAC1, or Fli1. The levels of endogenous p300 and HDAC1 were examined by Western blotting.
Figure 4.
TGF-β-induced histone H3 acetylation correlates with Fli1/HDAC1 dissociation and Ets1/p300 recruitment to the COL1A2 promoter. A.
Formaldehyde-cross-linked, moderately sheared chromatin was prepared from confluent quiescent fibroblasts treated with Trichostatin A (200 nM), anacardic acid (50 µM), or DMSO for 24 hours. The DNA fragments were immunoprecipitated using anti-acetylated histone H3 antibody, and the presence of the human α2(I) collagen (COL1A2) promoter fragments was detected using PCR. B. Formaldehyde-cross-linked, moderately sheared chromatin was prepared from foreskin fibroblasts untreated or treated with TGF-β1 (2.5 ng/ml) for 3 hours. The DNA fragments were immunoprecipitated using antibodies against Fli1, Ets1, p300, HDAC1, and acetylated histone H3. The presence of COL1A2 promoter fragments was detected using PCR.
Figure 5.
HDAC1 inhibits collagen gene expression in dermal fibroblasts. A.
Foreskin fibroblasts were transfected with the −772 COL1A2/CAT construct (2 µg), along with wild type Fli1 or the Fli1 K380R mutant (0.1 µg) and HDAC1 (0, 0.1, 0.3, or 0.5 µg) for 48 h. Values represent CAT activities relative to those of untreated cells (100 arbitrary units [AU]). The mean and SD from three separate experiments are shown. * p<0.05 versus control cells. ** p<0.05 versus cells transfected with wild type Fli1 only. # p<0.05 versus control cells. ## p<0.05 versus cells transfected with Fli1 K380R only. B. Confluent quiescent foreskin fibroblasts were treated with HDAC1 inhibitor or vehicle for 24 hours. Type I procollagen protein levels in whole cell lysates were determined by immunoblotting. A representative result of three independent experiments is shown. The band density was evaluated by densitometry. C. Under the same conditions, mRNA levels of the α1(I) collagen (COL1A1) gene were determined using reverse transcription quantitative real-time PCR. The graph represents -fold change in COL1A1 mRNA levels in comparison to unstimulated controls, which were arbitrarily set at 100. The mean and SD from three separate experiments are shown. * p<0.05 versus control cells treated with vehicle.
Figure 6.
TGF-β-induced remodeling of the transcription factor complex at the Ets binding site in the human COL1A2 promoter.
In quiescent cells, Fli1 forms a transcription repressor complex with p300 and HDAC1. HDAC1/p300 promotes deacetylation of Fli1, resulting in increased Fli1 DNA binding and a repressed chromatin state. After TGF-β stimulation, Fli1 phosphorylation by protein kinase C-δ induces disassembly of this transcription repressor complex and the acetylation of Fli1 by PCAF, leading to the loss of Fli1 DNA binding. Subsequently, the transcription activator complex consisting of Ets1 and p300 binds to the Ets binding site and activates transcription at least partially by promoting histone acetylation.