ABSTRACT
Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17ß-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.
Subject(s)
Breast Neoplasms/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic , Nucleosomes/metabolism , Proteomics , Response Elements/genetics , Breast Neoplasms/genetics , Chromatin Immunoprecipitation , DNA/genetics , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/genetics , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Estrogen Receptor alpha/genetics , Estrogens/pharmacology , Female , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , HeLa Cells , Humans , MCF-7 Cells , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Receptor Coactivator 3/genetics , Nuclear Receptor Coactivator 3/metabolism , Nucleosomes/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Promoter Regions, Genetic , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trans-Activators , Transcription, Genetic , Transcriptional ActivationABSTRACT
To evaluate whether alpha-smooth muscle actin (alpha-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of alpha-SMA, with that of lung fibroblasts (LFs), expressing high levels of alpha-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of alpha-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for alpha-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFbeta1 increased alpha-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFbeta-antagonizing agents reduced alpha-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with alpha-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with alpha-cardiac and beta- or gamma-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased alpha-SMA expression is sufficient to enhance fibroblast contractile activity.
Subject(s)
Actins/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Muscle, Smooth/metabolism , 3T3 Cells , Actins/genetics , Animals , Blotting, Western , Cell Size/drug effects , Cells, Cultured , Collagen/metabolism , Fibroblasts/drug effects , Gels , Mice , Microscopy, Fluorescence , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Silicon/metabolism , Transfection , Transforming Growth Factor beta/pharmacology , Up-RegulationABSTRACT
PURPOSE: Ionizing radiation has been shown to be a powerful inhibitor of neointimal hyperplasia following arterial injury in several animal models of post-percutaneous transluminal coronary angioplasty (post-PTCA) restenosis. This was previously shown to be associated with a reduction in smooth muscle cell (SMC) mitotic activity. This study evaluated the effect of intraarterial beta irradiation on the arterial wall SMC density and apoptosis. METHODS AND MATERIALS: Twenty-five carotid and 7 iliac arteries of hypercholesterolemic New Zealand white rabbits were injured using the Baumgartner technique. The impact of an 18 Gy beta radiation dose administered after balloon injury was studied and compared to a nonirradiated injured control group. The medial SMC density as well as the percentage of apoptotic cells were determined at 8 days, 21 days, and 6 weeks after injury using an automated computer-based software. Apoptotic cells were identified using in situ end-labeling of fragmented DNA. RESULTS: The values for medial apoptosis in control vs. irradiated arteries were: 0.014 +/- 0.023 vs. 0.23 +/- 0.28%, p = NS, at 8 days; 0.012 +/- 0.018 vs. 0.07 +/- 0.07%, p = 0.05, at 21 days; and 0 +/- 0 vs. 0.16 +/- 0.11%, p = 0.03, at 6 weeks. The overall incidence of medial apoptotic cells at all time points was 0.01 +/- 0.017 vs. 0.13 +/- 0.14% in controls and irradiated arteries respectively, p = 0.004. Medial SMC density was significantly decreased in irradiated arteries in comparison with controls (p < 0.01 at all time-points). CONCLUSIONS: Intraarterial beta irradiation stimulates medial SMC apoptosis in balloon-injured arteries. This, together with a decrease in SMC mitotic activity, contributes to a decrease in the arterial wall cellularity.