Lahm et al. 10.1073/pnas.0706487104. |
Fig. 7. Alignment of histone deacetylases (HDAC) class I and II sequences within the conserved HDAC domain. Motifs distinguishing class I and II HDACs are highlighted by boxes (Mot a and Mot b). Residues acting as Zn ligands, involved in substrate/inhibitor binding, or participating in the catalytic reaction are indicated by Z, S, or C, respectively, above the alignment. HDAC class assignment is indicated on the left. Motif b includes the conserved Y stabilizing the transition-state intermediate. Class IIa homologs like the ones found in D. melanogaster, D. pseudoobscura, C. elegans, or C. briggsae retain the Y. In vertebrate class IIa HDACs (exemplified by sequences from Homo sapiens), this residue is instead always substituted by H and Class IIa homologs from three ascidians have the Y substituted by F and the preceding D Zn ligand substituted by S. Sequences included in the alignment and corresponding NCBI GI identifiers are: hsHDAC11 (Homo sapiens HDAC11, 26394832); aaHDLP (Aquifex aeolicus HDLP corresponding to PDB entry 1C3R, 6137503); hsHDAC1 (Homo sapiens HDAC1, 13128860); hsHDAC2 (Homo sapiens HDAC2, 116284376); hsHDAC3 (Homo sapiens HDAC3, 13128862); hsHDAC8 (Homo sapiens HDAC8, 8923769); hsHDAC6_D1 (Homo sapiens HDAC6 domain 1, 10720024); hsHDAC6_D2 (Homo sapiens HDAC6 domain 2, 10720024); hsHDAC10 (Homo sapiens HDAC10, 20070354); hsHDAC4 (Homo sapiens HDAC4, 5174481); hsHDAC7 (Homo sapiens HDAC7, 13259522); hsHDAC9 (Homo sapiens HDAC7, 30795202); hsHDAC5 (Homo sapiens HDAC7, 62750347); dmHDAC4 (Drosophila melanogaster HDAC4, 24641718); dpHDAC4 (Drosophila pseudoobscura HDAC4, 54643587); ceHDA-4 (Caenorhabditis elegans HDAC4, 71982939); cbHDA-4 (Caenorhabditis briggsae HDAC4, 57012770); and abFB188 (Bordetella/Alcaligenes strain FB188 HDAH corresponding to PDB entry 1ZZ1, 83753890). Sequences of class IIa homolog of the ascidians Ciona intestinalis (ci), Ciona savignyi (cs), and Halocynthia roretzi (hr) shown in the alignment have been assembled using the following strategies: assembly of EST sequences GI 16853902,24629592,19478116,19467293,19486367,19480504,47757641, 19458053,19464306,24832292,16868008,16782318,16846314,16871165,24180654,1682 7588, 16856440,24480032,24259314,24833623,47758552,24579068,2488234,19478400, 48133587,48133086,48494720,19485608,19490329,24147367,24881030,24844060, 19494328,24893866,24885684,24893776,24882468,24887583,24856734,47816598, 24857049 (C. intestinalis); annotation of genomic sequence contigs AACT01057201, AACT01003964, AACT01009809, AACT01050682 (C. savignyi); assembly of cDNA sequence fragments GI 117756481,117771683,117766623 (H. roretzi). For C. intestinalis, where multiple sequences covering the region of interest were available, the substitutions of Y by F and of D by S (Zn ligand) were confirmed in all cases.
Fig. 8. Different phenotypes generated by substituting either the class I catalytic Y, or the corresponding class IIa H residue. Activity assays on WT (dashed bars) and the corresponding mutant (filled bars) HDAC-FLAG (HDAC-F) proteins were performed on pepH4-AcK16 substrate. (A) Enzymatic activities of WT and Y298F-mutated HDAC3-F proteins. (B) Western blot analysis of the HDAC3-F immunocomplexes assayed in (A) by using anti-FLAG (HDAC3-F), or anti-Gal4 (GAL4-DAD) antibodies. Forty nanomolar WT and mutant HDAC3 were analyzed. (C) Enzymatic activities of WT and Y303H-mutated HDAC1-FLAG proteins. (D) Western blot analysis of the HDAC1-F immunocomplexes assayed in C by using anti-FLAG (HDAC1-F), anti-Sin3A, anti-MTA2, anti-CoREST, or anti-HDAC2 antibodies. One hundred nanomolar WT and mutant HDAC1 were analyzed. (E) Enzymatic activities of WT and H976F-mutated HDAC4-F proteins on pepH4-AcK16 substrate. The H976F mutation failed to impair HDAC4-associated activity also in the contest of an N-terminally flag-tagged enzyme and on different acetylated substrates, such as 3[H]-acetylated core histones or the commercial Fluor de Lys substrate (not shown). (F) Western blot analysis of the HDAC4-F immunocomplexes assayed in E by using anti-FLAG (HDAC4-F), anti-HDAC3, or anti-N-CoR antibodies. One hundred nanomolar WT and mutant HDAC4 were analyzed.
Fig. 9. HDAC compound-binding assays. (A) UV cross-linking of HDAC3-FLAG and HDAC6-FLAG (10 nM) to the LAQ824 or apicidin derivatives. Linkerized/biotinylated compounds were tested at concentrations of 40 and 60 nM (HDAC3), or 40, 60, and 90 nM (HDAC6), and covalently cross-linked HDACs were analyzed by SDS/PAGE followed by ExtrAvidin Western blot. (B and C) Competitive binding experiments on LAQ824-HDAC4-FLAG complexes by using a panel of well known histone deacetylase inhibitor (HDACi). (B) Pull-down of HDAC4-FLAG (0.2 mM) on biotinylated LAQ824 (5 mM)-coated beads after preincubation with 50 mM of the indicated HDACi. A representative SDS/PAGE gel loaded with input (I), unbound (U), and bound (B) fractions and stained with Coomassie blue is shown. (C) UV cross-linking of HDAC4-FLAG to the LAQ824 derivative (or to the negative control derivative lacking the HDACis moiety) after preincubation with 10-, 100-, or 1,000-fold excess of the indicated HDACi. Ten nanomolar enzyme was preincubated with the different HDACis at .4, 4, or 40 mM at room temperature (RT) for 15 min before adding 40 nM LAQ824 ligand. An inactive HDACi analog was also included to control nonspecific competition. The HDAC4-FLAG fraction covalently cross-linked to the LAQ824 derivative was analyzed by SDS/PAGE followed by ExtrAvidin Western blot. Open apicidin (Open apic.) structure-activity relationships are described in Jones et al. [Jones P, Altamura S, Chakravarty PK, Cecchetti O, De Francesco R, Gallinari P, Ingenito R, Meinke PT, Petrocchi A, Rowley M, Scarpelli R, Serafini S, Steinkühler C (2006) Bioorg Med Chem Lett 16:5948-5952.]
Fig. 10. HDAC4 has a significantly lower enzymatic activity than class I HDACs. (A) Enzymatic activity of the HDAC4 catalytic domain produced in E. coli (HDAC4-CD) using the commercial (Biomol) assay. An enzyme concentration of 2 mM was needed to measure significant activity levels. Deacetylation was evaluated in the absence or presence of 10 mM apicidin (inactive) or LAQ824 (active). (B) Dose-response activity curves of HDAC1-FLAG (HDAC1-F, dots) and HDAC3-FLAG (HDAC3-F, triangles) using the commercial Biomol assay are shown for comparison. Notably, 5 nM class I enzymes show activities 10-fold (HDAC1) or 5-fold (HDAC3) higher than that measured with 2 mM HDAC4-CD.
Fig. 11. Acquisition of a "gain-of-function phenotype" by HDAC5 and HDAC7 H to Y mutants. (A) Comparison of the enzymatic activities exhibited by WT (open bars) and H1006Y-mutated (filled bars) HDAC5-FLAG proteins in the histone-based assay. Deacetylation was measured in the absence or presence of 300 nM HDACis (dashed bars), namely LAQ824 (+LAQ) or apicidin (+Ap). (B) Comparison of the enzymatic activities exhibited by WT (filled circles) and H843Y-mutated (open triangles) HDAC7-FLAG proteins using the commercial Biomol assay.
Fig. 12. Repression of MEF2-dependent transcription by WT and H976Y-mutated HDAC4-FLAG proteins. (A) One microgram of 4xMEF2-Luc was cotransfected into HeLa cells along with 1 mg of pCDNA3 (filled bar), or with increasing concentrations of WT pHDAC4-F (dashed bars), or H976Y-mutated pHDAC4-F (open bars). Total plasmid DNA amounts were normalized to 2 mg in all samples by adding pCDNA3 DNA. Fifty nanograms of pGL4.74 vector constitutively expressing Renilla luciferase were cotransfected for normalization. Twnety-four hours posttransfection, cells were harvested to evaluate both Firefly and Renilla luciferase activities, and normalized MEF2-Luc activity values were plotted. Average values of two independent experiments are shown with standard deviation bars. HDAC4-FLAG expression levels in cell lysates were controlled by Western blot using anti-FLAG monoclonal antibodies. Anti-GAPDH antibodies were included for normalization. (B) One microgram of 4xMEF2-Luc was cotransfected into HeLa cells together with 1 mg of pCDNA3 (filled bars), 1 mg of WT pHDAC4-F (dashed bars), or 1 mg of H976Ymutated pHDAC4-F (open bars). Treatments with LAQ824 (0.3 mM) or apicidin (1 mM) were carried out for 16 h before harvesting. In this case, luciferase activity values were normalized by total protein concentration. Luciferase activity values from transfections in the absence of any protein expression plasmid and of any HDACi treatment were arbitrarily set to 100%. Average values of three independent experiments are shown with standard deviation bars. HDAC4-FLAG expression levels were controlled as in A.
SI Methods
Plasmids.
Mammalian expression plasmids pHDAC3-F, pHDAC4-F, pHDAC5-F, and pHDAC7-F were obtained by standard PCR amplification of the HDAC coding sequences and insertion into a pcDNA3 vector (Invitrogen), upstream of a previously engineered FLAG coding sequence. pGAL4-DBD-NCoR DAD expression plasmid was constructed by PCR amplification of the murine N-CoR DNA sequence (nt 1207-1491) encoding a polypeptide homologous to the SMRT-DAD (22), and spanning N-CoR amino acids 403-497. This fragment was inserted into pcDNA3, downstream of a previously engineered GAL4DBD coding sequence. The prokaryotic pHis-HDAC4-CD expression vector was obtained by subcloning a DNA fragment encoding HDAC4 from T653 to L1084 downstream to the 6xHis-tag provided by the expression vector pET14b (Novagen). Mutated plasmids were derived from the corresponding WT expression vectors by using the QuickChange site-directed mutagenesis kit (Stratagene). All constructs were verified by DNA sequencing.Cell culture and transfections.
HEK293 and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen). Transfections were performed by using Lipofectamine (Invitrogen) according to the manufacturer's instructions. For further analysis, cells were harvested 24 h after transfection.Flag-tagged-HDACs expression in mammalian cells and affinity purification.
After transfection with pHDAC4, 5, 7-F, or cotransfection with pHDAC3-F and pGAL4NCoR-DAD expression vectors, HEK293 cells were collected in ice-cold PBS, homogenized, and sonicated in lysis buffer (20 mM Hepes, pH 7.9/0.25 mM EDTA/10% glycerol/300 mM NaCl/0.5% Nonidet P-40) containing 1 mM PMSF and Complete EDTA-free protease inhibitors mixture (Boehringer), followed by a 1-h incubation at 4°C. Soluble whole-cell extracts were obtained by centrifugation at 12,000 rpm in Sorval SS-34 rotor (Sorvall, Newton, CT) for 30 min at 4°C. Flagged HDAC complexes were immunoprecipitated on anti-FLAG M2 affinity gel (Sigma-Aldrich) and eluted in 50 mM Hepes (pH 7.4), 5% glycerol, 0.01% Triton X-100, and 100 mM NaCl in the presence of 100 mg/ml of 3x FLAG peptide (Sigma-Aldrich). Flag-tagged-HDAC concentrations were determined on Coomassie blue-stained SDS/PAGE gels by using a reference protein for quantification. The presence of GAL4 N-CoR DAD in the HDAC3-FLAG immunocomplexes was evaluated by Western blot analysis using anti-GAL4 DBD antibodies (Santa Cruz Biotechnology). Antibodies anti-HDAC2 and anti-MTA2 (Abcam), anti-CoREST (BD Biosciences), and anti-mSin3A (Affinity Bioreagents) were used for Western blot analysis of the HDAC1-FLAG-associated endogenous proteins.Anti-HDAC3 antibodies from Abcam and anti-N-CoR antibodies from Upstate Biotechnology were used for Western blot analysis of the HDAC4-FLAG-associated endogenous proteins.
HDAC4-CD purification from E. coli.
pHis-HDAC4-CD was transformed into E. coli BL21 Codon Plus (Invitrogen) cells and protein expression was induced upon addition of IPTG. After overnight incubation at 18°C, cells were disrupted in extraction buffer (25 mM Hepes, pH 7.5/30% glycerol/200 mM KCl/2 mM DTT/0.5% TritonX-100/Complete EDTA-free protease inhibitors mixture), and HDAC4-CD was purified to apparent homogeneity (Coomassie-stained SDS/PAGE) by Ni2+-chelating and anion-exchange (Resource Q) chromatography.Reporter gene assays.
The assays were performed with the luciferase reporter 4xMEF2-cFos-Luc containing four tandem copies of the MEF2-responsive element cloned upstream of the cFos minimal promoter (from -53 to + 51) to drive the expression of the firefly luciferase gene. Briefly, 1 mg of 4xMEF2-cFos-Luc DNA was transfected into 5 ´ 105 HeLa cells (in a well of a six-well plate), along with the indicated amounts of WT or mutant pHDAC4-F DNA. Total amounts of plasmid DNA were normalized to 2 mg in all samples by adding pCDNA3 DNA. In some experiments, pGL4.74 (hRluc/TK, Promega; 50 ng]-encoding Renilla luciferase under the control of HSV-TK promoter was used as coreporter for normalization. Twenty-four hours after transfection, cells were lysed, and luciferase activity values were determined by using the Dual-Luciferase Reporter assay system (Promega) and a TopCounter. In some experiments, cells were treated with HDACi for 16 h before harvesting. In this case luciferase activity values were normalized by total protein concentration. HDAC4-FLAG expression levels were controlled in the cell lysates by Western blot using anti-FLAG monoclonal antibodies (M2-AP; Sigma-Aldrich). Anti-GAPDH antibodies (Abcam) were used for normalization. Each transfection was performed at least two times.Synthesis of the trifluoroacetyl-lysine.
The trifluoroacetamide substrate tert-butyl {(1S)-1-{[(4-methyl-2-oxo-2H-chromen-7yl)amino]carbonyl}-5-[(trifluoroacetyl)amino]pentyl}carbamate was prepared as follows. To a solution of Boc-Lys-AMC (1 equivalent, BACHEM: I-1880) in DCM and Et3N (2 equivalents) at room temperature (RT) was added dropwise trifluoroacetic anhydride (1.2 equivalents). The resulting mixture was stirred at RT for 2 h and was then diluted with DCM and washed with saturated aqueous NaHCO3 solution and brine. The solution was dried (Na2SO4) and concentrated under reduced pressure while dry loading onto silica. The mixture was columned on silica eluting with 70-80% EtOAc/petroleum ether to yield the desired amide. 1H NMR (300 MHz, d6-DMSO) d: 10.40 (1H, br. s), 9.39 (1H, br. s), 7.76 (1H, d, J = 1.6 Hz), 7.72 (1H, d, J = 8.4 Hz), 7.48 (1H, d, J = 8.4 Hz), 7.10 (1H, d, J = 7.6 Hz), 6.62 (1H, s), 4.10-4.00 (1H, m), 3.21-3.10 (2H, m), 2.38 (3H, s), 1.70-1.55 (2H, m), 1.54-1.43 (2H, m), 1.40-1.25 (2H, m), 1.37 (9H, s). MS (ES) C23H28F3N3O6 requires: 499, found: 500 (M+H)+.