Specificity of Inhibition and Association of HDACs with Regions of c-<i>fos</i> and c-<i>jun</i>

<div><p>(A) Quiescent C3H 10T½ cells were treated with TSA (10 ng/ml) and HDAC inhibitor API or TPX for 15 min to 4 h. “C” indicates control (unstimulated). Acid-soluble proteins were extracted and separated on acid-urea gels. Western blots were carried out with anti-acetyl-H3 antibodies (panel i). A representative gel was stained with Coomassie to indicate protein loading (panel ii). Positions of histone isoforms are shown on the right of each panel, with zero being unmodified histone H3.</p> <p>(B) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with TSA (10 ng/ml) and HDAC inhibitor API or TPX for 15 min. Cells were then left unstimulated (C) or stimulated with TPA for 30 or 60 min. RNA was analysed by Northern blot.</p> <p>(C) Cross-linked chromatin fragments were prepared from untreated quiescent C3H 10T½ cells. Specific DNAs were immunoprecipitated with anti-HDAC1, anti-HDAC3, anti-HDAC4, or anti-HDAC6 antibodies. Recovered DNAs from antibody-bound fractions as well as total input DNA (Input) from released chromatin used for ChIP were analysed for the presence of c-<i>fos</i> (panels i and ii) and c-<i>jun</i> (panels iii and iv) gene sequences.</p></div

Acetylation and Methylation of Histone H3 TSA- and TPA-Treated Cells

<div><p>(A) Quiescent C3H 10T½ cells were treated with increasing concentrations of TSA (1, 10, or 500 ng/ml; 15 min to 4 h). “C” indicates control (unstimulated).</p> <p>(B) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with increasing concentrations of TSA (1, 10, or 500 ng/ml; 15 min). Cells were left unstimulated (C) or stimulated with TPA (15 to 60 min).</p> <p>(C) Quiescent C3H 10T½ cells were treated with TSA (10 or 500 ng/ml; 5 min to 4 h).</p> <p>Acid-soluble proteins were extracted and separated on acid-urea gels. Western blots were carried out with anti-acetyl-H3 ([A], panel i; [B], panel ii; [C], panel v), anti-phospho-H3 ([B], panel i), anti-phosphoacetyl-H3 ([B], panel iii), anti-monomethyl-K4 H3 ([C], panel i), anti-dimethyl-K4 H3 ([C], panel ii), anti-trimethyl-K4 H3 ([C], panel iii), or anti-dimethyl-K9 H3 ([C], panel iv) antibodies. An equivalent gel was stained with Coomassie to control for protein loading ([A], panel ii; [B], panel iv; [C], panel vi). Positions of histone isoforms are shown on the right of each panel, with zero being unmodified histone H3.</p></div

Effect of TSA Pre-Treatment on TPA-Stimulated MAP Kinase Activation and Transcription Factor Phosphorylation

<div><p>(A) Quiescent C3H 10T½ cells were treated with TSA (10 or 500 ng/ml; 5 min to 4 h). Positive controls for MAP kinase activation included ERK1/2 (TPA [T]; 10 min), JNK/SAPKs, and p38 (sAn; 30 to 60 min). “C” indicates control (unstimulated). Cell extracts were analysed by Western blotting with anti-ERK1/2, anti-phospho-p38, and anti–ACTIVE JNK antibodies. The mobility of ERKs is retarded on activation. Activation of p38 and JNK/SAPK results in phosphorylation. Note that anti–ACTIVE JNK also recognises activated ERK1/2 (lane 14).</p> <p>(B) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with TSA (10 or 500 ng/ml; 15 min). Cells were then left unstimulated (C) or stimulated with TPA for 5 to 30 min. Cell extracts were analysed by Western blotting with anti-ERK1/2 antibody.</p> <p>(C) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with TSA (500 ng/ml; 15 min). Cells were stimulated with TPA for 15 to 30 min. Cell extracts were analysed by Western blotting with anti-ATF-2 and anti-phospho-CREB antibodies. Phosphorylation of ATF-2 results in retarded mobility.</p></div

Histone H3 K4 Trimethylation and TSA Hypersensitivity at c-<i>fos</i> and c-<i>jun</i>

<div><p>(A and B) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells treated with TSA (10 ng/ml; 15 to 30 min) or stimulated with TPA (T; 30 min). “C” indicates control (unstimulated). Chromatin fragments were immunoprecipitated with anti-trimethyl-K4 H3 (3K4 IP) or anti-acetyl-H3 (AcH3 IP) antibodies.</p> <p>(C) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells untreated (−) or pre-treated with TSA (10 ng/ml; 15 min). Cells were left unstimulated (C) or stimulated with TPA (T; 30 min). Chromatin fragments were immunoprecipitated with anti-trimethyl-K4 H3 (3K4 IP) or anti-phosphoacetyl-H3 (PAcH3 IP) antibodies.</p> <p>DNA recovered from antibody-bound fractions as well as total input DNA (Input) from released chromatin used for ChIP was analysed for the presence of c-<i>fos</i> ([A]; [C], panels i and ii) and c-<i>jun</i> ([B]; [C], panels iii and iv) gene sequences. DNA immunoprecipitated with anti-acetyl-H3 antibody was diluted one in three, anti-trimethyl-K4 H3 was diluted one in 20, and anti-phosphoacetyl-H3 antibody was not diluted before PCR analyses.</p></div

Histone H3 K4 Trimethylation and Acetylation at Other Genes

<div><p>(A) Schematic diagram representing relative positions of regions of <i>GAPDH</i> and β<i>-globin</i> amplified by primer pairs used in the PCR step of ChIP assays. Exons are indicated by boxes, open for untranslated regions and filled for coding regions. Polyadenylation sites (pA) are indicated.</p> <p>(B) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells treated with TSA (10 ng/ml; 15 to 30 min) or stimulated with TPA (T; 30 min). “C” indicates control (unstimulated). Specific DNAs were immunoprecipitated with anti-trimethyl-K4 H3 (3K4 IP) or anti-acetyl-H3 (AcH3 IP) antibodies. Recovered DNAs from antibody-bound fractions as well as total input DNA (Input) from released chromatin used for ChIP were analysed for the presence of <i>GAPDH</i> ([B], panels i and ii) and β<i>-globin</i> ([B], panels iii and iv) gene sequences. DNA immunoprecipitated with anti-acetyl-H3 antibody was diluted one in three and anti-trimethyl-K4 H3 was diluted one in 20 before PCR analyses.</p></div

Three Histone H3 Modifications: Methylation, Acetylation, and Phosphorylation Are Targeted to the Same Nucleosome

<div><p>(A) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells treated with TSA (10 ng/ml; 15 to 30 min) or stimulated with TPA (T; 30 min). “C” indicates control (unstimulated). Specific DNAs were immunoprecipitated directly (1st IP; upper line) with anti-acetyl-H3 (AcH3) or anti-trimethyl-K4 H3 (3K4) before a second IP with anti-acetyl-H3 (2nd IP; lower line) to analyse chromatin in the anti-trimethyl-K4 H3 unbound or bound fraction (Unbound AcH3 and Bound AcH3, respectively). DNA immunoprecipitated with anti-acetyl-H3 antibody (1st or 2nd IP) was diluted one in three before PCR analyses.</p> <p>(B) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells pre-treated with TSA (10 ng/ml; 15 min). Cells were unstimulated (C) or stimulated with TPA (T; 30 min). Specific DNAs were immunoprecipitated directly (1st IP; upper line) with anti-phosphoacetyl-H3 (PAcH3) or anti-trimethyl-K4 H3 (3K4) before a second IP (2nd IP; lower line) to analyse chromatin in the unbound or bound fraction with anti-phosphoacetyl-H3 (Unbound PAcH3), or with anti-trimethyl-K4 H3 antibodies (Bound 3K4). DNA immunoprecipitated with anti-phosphoacetyl-H3 (1st or 2nd IP) was not diluted before PCR analyses. Recovered DNAs as well as total input DNA (Input) from released chromatin used for ChIP were analysed for the presence of c-<i>fos</i> ([A], panels i and ii; [B], panels i and ii) and c-<i>jun</i> ([A], panels iii and iv; [B], panels iii and iv) gene sequences.</p></div

Effect of TSA Treatment on Acetylation of Nucleosomes Associated with c-<i>fos</i> and c-<i>jun</i> Genes

<div><p>(A) Schematic diagram representing relative positions of regions of c-<i>fos</i> and c-<i>jun</i> amplified by primer pairs used in the PCR step of ChIP assays. Exons are indicated by boxes, open for untranslated regions and filled for coding regions. Polyadenylation sites (pA) are indicated.</p> <p>(B and C) Cross-linked chromatin fragments were prepared from quiescent C3H 10T½ cells treated with TSA (10 ng/ml; 15 min to 4 h). “C” indicates control (unstimulated). Specific DNAs were immunoprecipitated with anti-acetyl-H3 antibodies. Recovered DNAs from antibody-bound fractions (AcH3 IP) as well as total input DNA (Input) from released chromatin used for ChIP were analysed for the presence of c-<i>fos</i> (B) and c-<i>jun</i> (C) gene sequences. Controls for PCR included a DNA minus (−DNA) reaction and 5- to 20-ng loadings of input DNA to ensure all amplifications were within the linear range. PCR reactions were carried out in triplicate and gels quantified by phosphorimaging. Representative gels are shown.</p> <p>(D) Data are expressed graphically as average Bound/Input (± standard deviation). Note that to correct for the dilution factor applied to anti-acetyl-H3 immunoprecipitated DNA prior to PCR analyses, values obtained from quantification of PCR gels were multiplied by three.</p></div

Effect of TSA Pre-Treatment on c-<i>fos</i> and c-<i>jun</i> Induction

<div><p>(A) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with TSA (500 ng/ml) for 15 min (TSA 15′) or 4 h (TSA 4h). Cells were left unstimulated (C) or stimulated with TPA for 15 to 60 min, and RNA was analysed by Northern blot.</p> <p>(B) Quiescent C3H 10T½ cells were untreated, or pre-treated with TSA (500 ng/ml [TPA- and EGF-stimulated cells] or 10 ng/ml [bFGF-, serum-, sAn-stimulated cells]) for 15 min or 4 h. Cells were left unstimulated, or stimulated with TPA (T), EGF (E), bFGF (bF), serum (S), or sAn for 30 or 60 min.</p> <p>(C) Quiescent C3H 10T½ cells were untreated (−) or pre-treated with TSA (500 ng/ml) for 15 min (TSA 15′), 30 min (TSA 30′), 1 h (TSA 1h), 2 h (TSA 2h), 3 h (TSA 3h), or 4 h (TSA 4h). Cells were left unstimulated (C) or stimulated with TPA for 30 or 60 min.</p> <p>Northern blots in (B) and (C) were quantified by phosphorimaging, corrected for variations in loading using <i>GAPDH,</i> and expressed graphically. The normal response at 30 min (c-<i>fos</i>)/60 min (c-<i>jun</i>) was set to 0%, and inhibition/enhancement in response to TSA pre-treatment expressed as a percentage change relative to this value.</p></div