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Cited by 609Histone Deacetylase Inhibitors ResearchGenomics and Chromatin DynamicsSirtuins and Resveratrol in Medicine

Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

Open access
Lahm, A., Paolini, C., Pallaoro, M., Nardi, M. C., Jones, P., Neddermann, P., Sambucini, S., Bottomley, M. J. +6 more·2007·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.0706487104PMC PMC2077257
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Abstract

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nε2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this “gain of function phenotype” could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro . Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.

AI snapshot

Written from the paper’s full text.

TL;DR

Vertebrate class IIa HDACs carry a conserved active-site Tyr-to-His substitution that makes them very inefficient on canonical acetyl-lysine substrates, with most deacetylase activity in class IIa complexes coming from associated class I HDACs. A single His-to-Tyr mutation restores high catalytic activity, and trifluoroacetyl-lysine is identified as a class IIa-specific substrate in vitro.

Key findings

  • The catalytic Tyr conserved in other HDACs is replaced by His in vertebrate class IIa HDACs; mutating HDAC3 Y298 to H abolished activity on pepH4-AcK16, and HDAC1 Y303H reduced activity 5- to 8-fold.
  • HDAC4 catalytic domain purified from E. coli had measurable activity but was at least 1,000-fold lower than class I HDACs and 100-fold lower than the HDAC4-FLAG complex; it was inactive on pepH4-AcK16.
  • HDAC4 H976Y produced a gain-of-function phenotype: >20-fold increased activity for HDAC4-FLAG and >1,000-fold for HDAC4-CD, reaching levels comparable to class I enzymes; similar effects were seen for HDAC5 H1006Y and HDAC7 H843Y.
  • Trifluoroacetyl-lysine was identified as a class IIa-specific substrate in vitro, processed by HDAC4 full-length and catalytic domain and by three class IIa enzymes, but poorly by class I and class IIb HDACs.

Methods

  • HDAC4 was used as a representative class IIa model with four approaches: HDAC inhibitor binding/inhibition comparison, functional analysis of purified HDAC4 catalytic domain expressed in E. coli, structure-guided mutagenesis, and substrate screening.
  • Deacetylase assays used 3[H]acetyl-core histones purified from HeLa cells or fluorescent pepH4-AcK16, plus a commercial Fluor de Lys assay; HDAC inhibitor activities were measured by preincubation with enzymes before substrate addition.
  • Binding was assessed by pull-down with biotinylated LAQ824/apicidin derivatives and by UV cross-linking; sequence analysis used CLUSTALW/Genedoc and structural analysis used bacterial class II homolog PDB 1ZZ1.

Limitations

  • Stated: the lack of isotype/class-specific HDAC inhibitors limits distinguishing class IIa intrinsic activity from associated class I HDACs; attempts to obtain active recombinant class IIa HDACs in heterologous systems were unsuccessful, and it remains unknown whether this reflects missing cofactors or endogenous class I enzymes.
  • Stated: the precise role of HDAC4 catalytic activity in atypical biological contexts remains to be elucidated, and identification of additional biological activities/substrates dependent on class IIa H-associated activity requires further work.
  • Evident from the text: the study relies heavily on in vitro assays and ectopically expressed proteins, and no natural physiological class IIa-specific substrate is identified; the proposed specialized vertebrate class IIa activity is therefore inferred from synthetic substrate data.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1073/pnas.0706487104

Cites

36 of 36 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 14 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

    Trifluoroacetyl-lysine is a class IIa-specific substrate in vitro.

    “Finally, we have identified trif luoroacetyl-lysine as a class IIa-specific substrate in vitro.”
    Quote in the paperSubstantial claim
  • Findingown data

    The Y298H substitution in HDAC3 does not affect its association with N-CoR DAD.

    “However, the Y298H substitution in HDAC3 did not affect its association with N-CoR DAD (Fig. 2B), suggesting that the integrity of the catalytic domain was preserved.”
    Quote in the paperSubstantial claim
  • Findingown data

    Replacement of Y with H in HDAC3 produces a loss-of-function phenotype as severe as replacement with F.

    “Furthermore, replacement of Y with H produced a loss-of-function phenotype as severe as that caused by replacement with F (SI Fig. 8A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Replacing the active site Y with H in HDAC3 (Y298H) abolishes its enzymatic activity on a histone H4-derived peptide containing a single acetyl-lysine (pepH4-AcK16).

    “As predicted, replacing the active site Y with H was sufficient to abolish the enzymatic activity on a histone H4-derived peptide containing a single acetyl-lysine (pepH4-AcK16) (Fig. 2A).”
    Quote in the paperSubstantial claim
  • Findingown data

    Mutating Y-303 into H in HDAC1-FLAG reduces HDAC1 deacetylase activity 5- to 8-fold.

    “As shown in SI Fig. 8C, the mutation actually reduced HDAC1 deacetylase activity 5-to 8-fold.”
    Quote in the paperSubstantial claim
  • May combine two claims. 23
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 4

  • May combine two claims.

    Findingown data

    In vertebrate class IIa HDACs, the catalytic Y residue is replaced by H, whereas other functional residues are virtually always conserved.

    “In the vertebrate class IIa HDACs, the catalytic Y is replaced by an H, a conservative substitution, whereas other functional residues are virtually always conserved (SI Fig. 7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The recombinant HDAC4 catalytic domain (HDAC4-CD) is efficiently pulled down on biotinylated LAQ824 beads, suggesting that the structural integrity of the catalytic domain is preserved.

    “This protein was efficiently pulled-down on biotinylated LAQ824 beads (Fig. 4A), suggesting that the structural integrity of the catalytic domain was preserved in the recombinant truncated mutant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LAQ824 and apicidin have similar effects in the transcriptional repression assay, in contrast to their different inhibitory potencies and binding affinities in vitro.

    “However, LAQ824 and apicidin had similar effects in this assay in contrast to the different inhibitory potencies and binding affinities shown in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HDAC5 H1006Y and HDAC7 H843Y mutants purified from mammalian cells exhibit strongly increased activity compared with WT and are inhibited by LAQ824 but resistant to apicidin.

    “Again Y mutants exhibited strongly increased activity compared with WT and, like the HDAC4 mutant, could be inhibited by LAQ824 but were resistant to apicidin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pull-down of HDAC4-CD on biotinylated LAQ824 beads is significantly reduced by preincubation with excess free LAQ824, whereas apicidin and MS275 do not compete.

    “Pull-down was significantly reduced by preincubation with an excess of free LAQ824, whereas apicidin and MS275 did not show competition, thus confirming and directly validating the results obtained with ectopic HDAC4-FLAG.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    H976Y-substituted HDAC4 enzymes are sensitive only to LAQ824 and are highly resistant to inhibition by MS-275, apicidin, and its open derivative.

    “Different than WT HDAC4-FLAG, Y mutants were sensitive only to the inhibitory effect of LAQ824, but they were highly resistant to inhibition by compounds belonging to different chemical classes (MS-275, apicidin, and its open derivative), all of which are active on class I HDACs (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HDAC4-CD is completely inactive on pepH4-AcK16 and on a number of acetylated peptides grafted from nonhistonic HDAC substrates.

    “In addition, HDAC4-CD was completely inactive on pepH4-AcK16 and on a number of acetylated peptides grafted from nonhistonic HDAC substrates (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Class IIa HDACs are not proficient enzymes on acetyl-lysines and are very inefficient on standard substrates.

    “Having ascertained that class IIa HDACs are not proficient enzymes on acetyl-lysines, we sought alternative in vitro substrates.”
    • Also stated in: § HDAC4 Compound Binding Assays. · boosted
      “Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Trifluoroacetyl-lysine is actively and specifically processed by three different class IIa enzymes, whereas it is a poor substrate for both class I and class IIb HDACs.

    “Moreover, this compound was actively and specifically processed by three different class IIa enzymes, whereas it was a poor substrate for both class I and class IIb HDACs (Fig. 6).”
    • Also stated in: § Discussion · plain
      “We have identified a class IIa-specific synthetic substrate, trifluoroacetyl-lysine.”
    • Also stated in: § HDAC4 Compound Binding Assays. · plain
      “Trifluoroacetyl-lysine is a class IIa-specific substrate in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    HDAC inhibitors LAQ824 and apicidin are unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay.

    “We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ascidian class IIa sequences lack one of the conserved Zn ligands, a D is replaced by an S.

    “Interestingly, ascidian class IIa sequences also lack one of the conserved Zn ligands, a D is replaced by an S (SI Fig. 7).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    In class IIa HDACs, three different residues (Y, H, and F) are observed at the catalytic position, and it is unlikely that a concomitant conformational change occurs for each residue.

    “In class IIa HDACs, three different residues (Y, H, and F) are observed at this position and, given the particular structural context, it is unlikely that for each residue a concomitant conformational change would occur.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The HDAC4 catalytic domain is properly folded under the experimental conditions, as shown by the fact that a single amino acid substitution (H976Y) is sufficient to confer high enzymatic activity.

    “This experimental evidence also proved the HDAC4 catalytic domain to be properly folded under our experimental conditions, because a single amino acid substitution was sufficient to confer high enzymatic activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The HDAC4-CD H976Y mutant shows an inhibition profile identical to that of the full-length HDAC4-FLAG mutant, suggesting that the observed selectivity is directly related to the catalytic domain and does not depend on additional N-terminal regions.

    “Furthermore, the HDAC4-CD mutant showed an inhibition profile identical to that of the full-length HDAC4-FLAG mutant, suggesting that the observed selectivity is directly related to the catalytic domain and does not depend on additional N-terminal regions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    HDAC4 does not contribute significantly to the deacetylase activity of the HDAC4 complex, or does so only to a marginal extent.

    “Together, these results demonstrate that HDAC4 intrinsic catalytic activity is low on canonical acetylated substrates and does not contribute significantly to the overall activity observed in purified ectopic HDAC4 complexes.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

    All class IIa members expressed in mammalian cells recruit class I HDACs, which possess high deacetylase activities both in vitro and in cells.

    “All class IIa members expressed in mammalian cells are known to recruit class I HDACs, which possess high deacetylase activities both in vitro and in cells (18)(19)(20).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites (18)(19)(20)

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Compounds that did not inhibit the H976Y-substituted HDAC4 enzymes were those that did not bind WT HDAC4, implying that the mutant proteins retain the WT selectivity of interactions.

    “Remarkably, compounds that did not inhibit the H976Ysubstituted enzymes were those that did not bind WT HDAC4, thus implying that the mutant proteins retain the WT selectivity of interactions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Class IIa HDACs are potent and selective transcriptional repressors.

    “It is well documented that class IIa HDACs are potent and selective transcriptional repressors (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The finding that class IIa HDAC Y mutants are proficient was confirmed in the isolated catalytic domain expressed in a prokaryotic system and in full-length enzymes expressed in mammalian cells, including HDAC5 and -7.

    “This finding was confirmed both in the isolated catalytic domain expressed in a prokaryotic system as well as for the full-length enzymes expressed in mammalian cells, including HDAC5 and -7.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Class IIa HDACs have intrinsically different catalytic activities in two groups: canonical HDAC activity in invertebrates and a more specialized enzymatic activity in vertebrates.

    “The emergent theme predicts class IIa HDACs with intrinsically different catalytic activities for the two groups: (i) canonical HDAC activity in invertebrates and (ii) a more specialized enzymatic activity in vertebrates.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The catalytic mechanism of HDACs is conserved across the enzymes and species considered.

  • May combine two claims.

    Interpretationreasoning

    The presence of the trifluoro group destabilizes the amide bond and favors the reaction even in the absence of transition-state stabilization.

    “Mechanistically, the presence of the trifluoro group should destabilize the amide bond and thus favor the reaction even in the absence of transition-state stabilization, as postulated for the His-containing class IIa HDACs.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The catalytic mechanism of HDACs is conserved across the enzymes and species considered.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Mean values of at least two independent assays are reported.

    “Mean values of at least two independent assays are reported.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Our MEF2 reporter gene data exclude HDAC4 catalytic activity as responsible for transcriptional repression and point to involvement of class I enzymes equally sensitive to inhibition by both LAQ824 and apicidin.

    “Our MEF2 reporter gene data are in agreement with these findings, excluding HDAC4 catalytic activity for being responsible for transcriptional repression and pointing to the involvement of class I enzymes that are equally sensitive to the inhibition by both LAQ824 and apicidin.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationown data

    Class IIa HDACs should be considered a distinct class of therapeutic targets, concerning both their natural substrates and selective mechanisms of inhibition.

    “Finally, our data also imply that class IIa HDACs should be considered a distinct class of therapeutic targets, concerning both their natural substrates and selective mechanisms of inhibition.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Vertebrate class IIa HDACs have natural substrates that are not canonical acetyl-lysines.
    • Rests on an assumption: In vitro substrate specificity of class IIa HDACs reflects their in vivo biological function.

  • May combine two claims.

    Premisecitation

    Sirt1 and HDAC3, but not HDAC4 or -5, efficiently deacetylate MEF2D, contributing to MEF2D-dependent transcriptional repression.

    “More recently, it has been shown that both Sirt1 (28) and HDAC3 (29), and not HDAC4 or -5, efficiently deacetylate MEF2D, thus contributing to MEF2Ddependent transcriptional repression.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [28] source

      Not checked in the free run.

    • cites [29] source

      Not checked in the free run.

25 of 25

  • The authors state this as an assumption.

    The catalytic mechanism of HDACs is conserved across the enzymes and species considered.

    “Based on our results, the presence of an F should be incompatible with activity, provided that the catalytic mechanism is conserved.”

  • The paper relies on this without support.

    Vertebrate class IIa HDACs have natural substrates that are not canonical acetyl-lysines.

    “Future work will undoubtedly identify these yet unknown physiological substrates and further reveal the already complex biological activities of these enzymes.”

  • The paper relies on this without support.

    In vitro substrate specificity of class IIa HDACs reflects their in vivo biological function.

    “our results point to a previously unknown, specific catalytic activity of vertebrate class IIa HDACs.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Class IIa enzymes, HDAC3, HDAC8, and HDAC11 removed the non-physiological Ktfa substrate.

    “Class IIa enzymes (Lahm et al., 2007) as well as HDAC3 (Madsen and Olsen, 2012a), HDAC8 (Bradner et al., 2010), and HDAC11 (Inks et al., 2012) removed the physiologically non-relevant trifluoroacetamide (Ktfa).”

    From Histone deacetylase 11 is an ε-N-Myristoyllysine hydrolase (2018) · cites this paper as Lahm et al., 2007

  • In vertebrates, class IIa HDACs have limited catalytic activity due to replacement of tyrosine with histidine in the enzymatic pocket.

    “In vertebrates, this subfamily is characterized by limited catalytic activity, mainly due to the replacement of a tyrosine (Y) with a histidine (H) residue in the enzymatic pocket (Lahm et al., 2007).”

    From HDACs and the epigenetic plasticity of cancer cells: target the complexity (2022) · cites this paper as Lahm et al., 2007

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Claim of universal Tyr conservation overlooks ascidian class IIa homologs

    Minor

    The first statement says Tyr is conserved in every HDAC outside vertebrate class IIa enzymes. The paper later identifies ascidian class IIa homologs with Phe at that position; ascidians are not vertebrates.

1 of 1

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Authors
Lahm, A., Paolini, C., Pallaoro, M., Nardi, M. C., Jones, P., Neddermann, P., Sambucini, S., Bottomley, M. J., Lo Surdo, P., Carfı́, A., Koch, U., De Francesco, R., Steinkühler, C., & Gallinari, P.
Affiliations
  • The University of Texas Southwestern Medical Center (US)
  • Istituto di Ricerche di Biologia Molecolare, I.R.B.M., P. Angeletti, 00040 Pomezia, Italy
Corresponding author
Gallinari, P.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2007-10-30
Volume
104
Issue
44
Pages
17335-17340
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.0706487104
PMC
PMC2077257
OpenAlex
W2152631096
Cited by
566
References
36
Citations by year
2026: 19 · 2025: 29 · 2024: 40 · 2023: 15 · 2022: 32 · 2021: 33 · 2020: 32 · 2019: 37 · 2018: 31 · 2017: 29 · 2016: 31 · 2015: 44 · 2014: 29 · 2013: 39 · 2012: 34
FWCI
9.53
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Histone Deacetylase Inhibitors Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Geriatrics and Gerontology › Sirtuins and Resveratrol in Medicine
Keywords
class IIa histone deacetylases, HDAC4, HDAC5, HDAC7, HDAC9, catalytic efficiency, histone deacetylase inhibitors, class I HDACs, catalytic tyrosine, enzyme evolution, substrate specificity
MeSH terms
Amino Acid Sequence, Animals, Binding Sites, Catalysis, Enzyme Activation, HeLa Cells, Histidine, Histone Deacetylases, Humans, Models, Molecular, Mutation, Substrate Specificity, Urochordata, Vertebrates, Protein Structure, Tertiary

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