A family of human histone deacetylases related to yeast HDA1.
To identify new mammalian histone deacetylases, we performed sequence database searches with BLAST and PSI-BLAST (1
). Using the amino acid sequence of yeast HDA1 as the bait, we found several human cDNA and genomic clones encoding polypeptides with significant sequence similarity to the catalytic domain of HDA1. Figure A shows the schematic representation of these novel polypeptides. Most of these clones were isolated in DNA sequencing projects, whereas HDAC5 was also isolated as a clone coding a human colon cancer antigen recognized by an autologous antibody (37
). Available sequence data indicated that HDAC4, -5, and -7 are homologous, with their C-terminal parts similar to the catalytic domain of HDA1 (Fig. A and B). Sequence alignment of the N-terminal domains of HDAC4, -5, and -7N is shown in Fig. C. HDAC6 possesses two homologous regions similar to the catalytic domain of HDA1, and a cysteine/histidine-rich domain located at its C-terminal part (Fig. A and B). The putative catalytic domains of HDAC4, -5, and -6 are more similar to yeast HDA1 (sequence identity of 35%) than to human HDAC1, -2, and -3 (sequence identity of 26%), suggesting that HDAC4, -5, and -6 and probably HDAC7 constitute a new subfamily of human histone deacetylases, with HDAC4, HDAC5, and probably HDAC7 more similar to each other than to HDAC6. Since HDAC4 was identified first and its full-length cDNA was available, we chose to characterize it further.
FIG. 1 Comparison of HDAC4-7 with HDA1. (A) Schematic representation of HDA1 and HDAC4 to -7. The N terminus of HDAC5 is incomplete, as are both termini of HDAC7. HDAC7N may be an alternatively spliced variant of HDAC7. The conserved deacetylase domains are (more ...)
To determine tissue distribution of HDAC4, Northern blot analyses were performed. These analyses indicated that HDAC4 is expressed in skeletal muscle, brain, leukocyte, colon, small intestine, and ovary but not in liver, lung, and placenta (Fig. ). To map the chromosomal localization of the HDAC4 gene, FISH analyses were performed. These analyses revealed that the HDAC4 gene is located at chromosome band 2q37.2 (Fig. ). Abnormalities in this region have been implicated in developmental delay and predisposition to certain cancers (8
). Moreover, this band has been found to contain a cellular senescence gene (52
FIG. 2 Expression of HDAC4 in various adult human tissues. Poly(A) RNA blots (Clontech; 2 μg/lane) were probed with an HDAC4 cDNA fragment derived from the 3′ untranslated region (top). As a loading control, the same blots were reprobed with (more ...)
FIG. 3 Chromosomal localization of the HDAC4 gene. Left, FISH signals detected at chromosome band 2q37.2, indicated by an arrow; right, the same mitotic cell stained with DAPI (4′,6-diamidino-2-phenylindole) to identify chromosomes. Human blood lymphocytes (more ...) Histone deacetylase activity of HDAC4.
To determine the histone deacetylase activity of HDAC4, Flag-tagged HDAC4 and deletion mutants dm1, -2, and -3 (Fig. A) were expressed in 293T cells and subject to histone deacetylase assays. As shown in Fig. B, affinity-purified HDAC4 efficiently deacetylated [3H]acetyl-histones. Mutant dm1 had activity 2.9-fold higher than that of full-length HDAC4. Whereas dm2 had minimal activity, dm3 was slightly more active than dm1, suggesting that dm3 contains a deacetylase domain. This is consistent with the observation that the HDA1-related domain of HDAC4 is located at its C-terminal part (Fig. A).
FIG. 4 Characterization of histone deacetylase activity of HDAC4. (A) Schematic representation of HDAC4 and its mutants used for deacetylase assays. The letters “HH” denote histidines 802 and 803, which may be essential for deacetylase activity. (more ...)
To establish that the observed deacetylase activity is intrinsic to HDAC4 (but not due to any associated proteins), we prepared mutants with histidines 802 and 803 replaced with lysine and leucine, respectively (Fig. A, H803L and dm1/H803L). Histidine residues at equivalent positions have been found to be important for the deacetylase activity of HDAC1 and RPD3 (18
). Compared with HDAC4 and dm1, both mutants had much lower deacetylase activity (Fig. B), suggesting that HDAC4 has intrinsic deacetylase activity and the histidine residues are important for the enzymatic activity.
To examine the effects of deacetylase inhibitors, we determined the deacetylase activity of dm3 in the presence of various concentrations of TSA or sodium butyrate. As shown in Fig. C, TSA dramatically inhibited the activity of dm3, with a 50% inhibitory concentration of 5 nM, whereas sodium butyrate (up to 5 mM) had much smaller effects. HDAC1 and HDAC3 are more sensitive to sodium butyrate than HDAC4 (10
Mutants dm1 and dm3 were also expressed in Sf9 cells, using the baculovirus expression system. Proteins prepared this way had activity inversely proportional to their expression levels. Even the most active preparations possessed much lower activity than those obtained from 293T cells (data not shown), suggesting that an elusive factor(s) required for deacetylase activity may not present in sufficient quantities in insect cells.
Tethered HDAC4 functions as a repressor.
The possession of intrinsic deacetylase activity by HDAC4 suggests that it may be involved in transcriptional regulation. To test this hypothesis, we first investigated if HDAC4 functions as a repressor when artificially tethered to a promoter. For this purpose, a mammalian vector was constructed to express HDAC4 fused to the Gal4 DNA-binding domain and tested by cotransfection assays with the Gal4-tk-Luc reporter (Fig. A) in NIH 3T3 cells. As shown in Fig. B, while the Gal4 DNA-binding domain itself activated transcription 2-fold, GAL4-HDAC4 repressed transcription 14-fold. To delineate the repression domain(s), mammalian vectors were constructed to express various HDAC4 mutants fused to the Gal4 DNA-binding domain. HDAC4 mutants tested include dm1 to -3 (Fig. A), dm4 (residues 1 to 208), and dm5 (residues 1 to 114). As shown in Fig. B, similar to Gal4-HDAC4, Gal4-dm1 repressed transcription 11-fold. While Gal4-dm2 had minimal effects (~2-fold), Gal4-dm3 repressed transcription 83-fold. In contrast, Gal4-dm3 had a much smaller repressive effect on the tk-Luc reporter (1.8-fold [data not shown]). Western analyses with an anti-Gal4 antibody indicated that Gal4-HDAC4 and Gal4-dm1 to -5 were indeed expressed (Fig. C). All of these results suggest that dm3 contains an active, strong repression domain. Unexpectedly, Gal4-dm4 repressed transcription 14-fold whereas both Gal4-dm2 and Gal4-dm5 had minimal effects (Fig. B), suggesting that residues 1 to 208 of HDAC4 constitute another repression domain.
FIG. 5 Tethered HDAC4 represses transcription. (A) Schematic representation of the luciferase reporter Gal4-tk-Luc. Upstream from the tk core promoter (−152 to +50) are five copies of the Gal4-binding site. (B) Repression of Gal4-tk-Luc by HDAC4 (more ...)
The repression observed with dm3 is stronger than that reported for HDAC1, -2, and -3 (59
). To assess if the repression by Gal4-dm3 is cell line dependent, we performed similar transfection assays in 293T cells. As shown in Fig. D, Gal4-dm3 repressed Gal4-tk-Luc reporter activity in these cells in a dose-dependent manner. Since repression mediated by HDAC1 was found to be promoter dependent (30
), we assessed if Gal4-dm3 is able to repress reporters containing other core promoters. For this purpose, transfection assays were performed with TATA-containing (Gal4-AdML-Luc and Gal4-SV40-Luc) as well as TATA-less (Gal4-CD4-Luc) reporters. As shown in Fig. D, Gal4-dm3 was able to repress transcription of all of these reporters. Taken together, these results suggest that once tethered to a promoter, the deacetylase domain of HDAC4 functions as a transcriptional repressor.
Requirement of HDAC4 deacetylase activity for repression.
The repression observed with HDAC4 could be due to deacetylation mediated by HDAC4 and/or to association with a repressor(s). This prompted us to examine whether the intrinsic deacetylase activity of HDAC4 is important for the observed repression. Since TSA inhibited deacetylase activity of HDAC4 (Fig. C), we determined effects of TSA on HDAC4-mediated repression. TSA only partially relieved repression mediated by Gal4-HDAC4 and Gal4-dm1 (Fig. B). TSA had a much more dramatic effect on the repression mediated by Gal4-dm3 (Fig. B), suggesting that histone deacetylase activity is important for the repression observed with Gal4-dm3. Substitution of histidines 802 and 803 reduced repression by Gal4-dm1, and TSA had no effects on residual repression observed with Gal4-dm1/H803L (Fig. B; compare Gal4-dm1 and Gal4-dm1/H803L). TSA did not relieve repression mediated by Gal4-dm4 (Fig. B). Taken together, these results suggest that while the histone deacetylase activity of HDAC4 is important for its repression function, mechanisms independent of deacetylation are also involved.
HDAC4 does not directly bind to DNA.
Since promoter tethering of HDAC4 leads to transcriptional repression, we next asked how HDAC4 is recruited to promoters in vivo. One possibility is that HDAC4 possesses intrinsic DNA-binding ability. Sequence-specific DNA-binding proteins can, although with lower affinity, bind to nonspecific DNA. To address if HDAC4 directly binds to DNA, we performed a DNA-binding assay to determine if HDAC4 could nonspecifically bind to fish sperm DNA (17
). This assay revealed that Flag-HDAC4 immobilized on M2-agarose could not retain a significantly higher amount of DNA than M2-agarose itself (data not shown). Therefore, HDAC4 does not have intrinsic DNA-binding ability.
HDAC4 physically interacts with MEF2 transcription factors.
Since HDAC4 does not bind to DNA by itself, we reasoned that other transcription factors might mediate the recruitment of HDAC4 to promoters. To identify such target transcription factors, we tested several active repressors, including human Groucho homolog TLE1 (12
), zinc finger oncoprotein Evi1 (3
), Polycomb-group protein EZH2 (28
), and adenovirus protein E1B (61
). Protein-protein interaction studies and reporter gene assays indicated that none of these repressors interact with HDAC4 (data not shown).
A novel Xenopus laevis
repressor protein, termed MITR (GenBank accession no. Z97214
; reference 47
), was identified as an interaction partner for the Xenopus
myocyte enhancer-binding factors SL-1 and -2. Xenopus
MITR is a homolog of HDAC7N (sequence identity, 59%; similarity, 67%). As illustrated in Fig. A, HDAC7N is composed of two regions, the N-terminal part of which shows significant sequence similarity to HDAC4 (sequence identity, 46%; similarity, 58%). In light of these observations, we tested if HDAC4 interacts with human MEF2 transcription factors.
To examine in vivo interaction between HDAC4 and MEF2s, we performed immunoprecipitation experiments in which HDAC4 (Flag tagged) and/or MEF2C expression plasmids were cotransfected into 293T cells, and extracts prepared from the transfected cells were subjected to immunoprecipitation with anti-Flag M2-agarose. Eluted immunocomplexes were subjected to Western blotting analyses with anti-Flag and anti-MEF2C antibodies. As shown in Fig. A, MEF2C specifically precipitated with Flag-tagged HDAC4 (lanes 1 to 4). Similar immunoprecipitation experiments revealed that HDAC4 precipitated with endogenous MEF2D (lanes 6 to 8). These results indicate that HDAC4 interacts with MEF2C and MEF2D in vivo.
FIG. 6 HDAC4 interacts with MEF2 in vivo and in vitro. (A) Immunoprecipitation of HDAC4 with MEF2C (lanes 1 to 5) or MEF2D (lanes 6 to 9). Flag-tagged HDAC4 (lanes 1 to 4 and 7) or dm4 (lanes 5 and 9) was expressed with (lanes 2, 4, and 5) or without (lanes (more ...)
These immunoprecipitation data also suggest that conserved regions of MEF2C and MEF2D mediate their interaction with HDAC4. Since the N-terminal regions of MEF2C and MEF2D contain the MADS-box and MEF2-specific domains and are the most conserved, we next examined whether the MEF2C mutant M178 could interact with HDAC4 (Fig. B). For this, M178 was expressed in E. coli as a fusion with MBP and used for in vitro pull-down assays. As shown in Fig. C, M178 specifically interacted with HDAC4 (lanes 1 to 3). To delineate regions of HDAC4 required for such interaction, we used a series of HDAC4 mutants (Fig. E). M178 interacted with dm1 (Fig. C, lanes 4 to 6) and less strongly with dm6 (lanes 7 to 9). By contrast, M178 did not interact with dm7 to -9 (lanes 10 to 18), suggesting that residues 118 to 188 of HDAC4 are essential for interaction with M178. Consistent with this contention, dm2 but not dm3 interacted with M178 (Fig. D, lanes 1 to 6). To further map the MEF2 interaction domain, dm4 and dm5 were tested. Unlike dm5, dm4 interacted with M178 (lanes 7 to 12), suggesting that residues 118 to 208 of HDAC4 are essential for interacting with M178. To determine whether these residues are sufficient, dm10 was used (Fig. E). This mutant was found to interact with M178 (Fig. D, lanes 13 to 15), confirming that residues 118 to 208 of HDAC4 are sufficient for interaction with MEF2C. Furthermore, in immunoprecipitation experiments, dm4 was found to interact with MEF2C (Fig. A, lane 5) or MEF2D (lane 9) in vivo. Taken together, these results indicate that residues 118 to 208 of HDAC4 contain a MEF2 interaction domain (Fig. E).
HDAC4 represses MEF2C-dependent transcription.
To explore the functional relevance of the observed physical interaction between HDAC4 and MEF2C, we constructed a luciferase reporter containing a MEF2-binding site (MEF2-E4-Luc [Fig. A]). This reporter was transfected into NIH 3T3 cells with or without expression plasmids for HDAC4 and/or MEF2C. As expected, MEF2C activated the reporter (Fig. B). While HDAC4 itself had minimal effects on the reporter activity in the absence of cotransfected MEF2C, HDAC4 repressed MEF2C-dependent transcription in a dose-dependent manner. The HDAC4 mutant dm7, which lacks a MEF2-binding site, had a much smaller effect. Since recruitment of HDAC4 by MEF2C repressed the reporter activity below the control level, HDAC4 may not be only inhibitory to the activation function of MEF2C. To substantiate this point, the MEF2C mutant M178 was tested. This mutant only weakly stimulated the reporter activity since it lacks the MEF2C activation domain located at its C-terminal part (Fig. C). In a dose-dependent manner, HDAC4 repressed the reporter activity below the control level. On the other hand, dm7 had minimal effects. Western blotting analyses revealed that HDAC4 and dm7 were expressed at similar levels (data not shown). Taken together, these results suggest that MEF2C recruits HDAC4 to repress transcription.
FIG. 7 HDAC4 represses transcription in a MEF2C-dependent manner. (A) Schematic representation of the reporter MEF2-E4-Luc, which contains one copy of the MEF2-binding site upstream from the adenovirus E4 core promoter (−34 to +34) and the luciferase (more ...) HDAC4 cooperates with MEF2C to inhibit c-jun promoter activity.
Next we wished to examine a native promoter containing a MEF2-binding site. In nonmuscle cells, MEF2C regulates the expression of the proto-oncogene c-jun
). Therefore, we tested the reporter pJLuc (Fig. A), which contains the c-jun
promoter upstream from the luciferase gene (Fig. A; reference 16
FIG. 8 HDAC4 and MEF2C cooperatively regulate c-jun promoter activity. (A) Schematic representation of the reporter pJLuc, which contains positions −225 to +150 of the c-jun promoter upstream of the luciferase coding sequence. (B) HDAC4 activates (more ...)
First, the expression plasmid for HDAC4 was cotransfected with this reporter to verify that HDAC4 does not regulate the promoter in the absence of cotransfected MEF2C. Unexpectedly, HDAC4 increased the reporter activity eightfold (Fig. B). To localize regions of HDAC4 involved in such activation, several deletion mutants were tested. While mutants dm2 to -5 had minimal effects, dm1 and dm7 activated the reporter 4- and 10-fold, respectively. Since dm7 lacks MEF2C-binding ability (Fig. E), HDAC4-mediated activation of pJLuc may be independent of MEF2C. Substitution of histidines 802 and 803 greatly diminished the activation ability of both HDAC4 and dm1 (Fig. B; compare the mutants H803L and dm1/H803L with HDAC4 and dm1, respectively), suggesting that the histone deacetylase activity of HDAC4 is important for activation of the c-jun promoter.
We then investigated the effects of MEF2C on the reporter pJLuc. As expected, transfection of MEF2C activated the expression of this reporter 15-fold (Fig. C). Cotransfection of HDAC4 repressed the activation mediated by MEF2C below the control level (Fig. C), raising an intriguing regulation scheme: transfected HDAC4 and MEF2C individually activate but together repress c-jun promoter activity. To determine which region of HDAC4 is required for this repression, we tested HDAC4 deletion mutants. Mutant dm1 repressed transcription 28-fold, whereas dm2 and dm3 had minimal effects (Fig. C and D), suggesting that both the deacetylase domain and residues 118 to 626 are required for dm1 to repress MEF2C-dependent transcription. dm7 repressed the reporter activity less efficiently than dm1 (Fig. C and D). Since dm7 lacks the MEF2C-binding domain (Fig. E), these results suggest that the MEF2C interaction domain is important for dm1 to repress transcription of the reporter pJLuc.
Mutant dm4 repressed transcription 49-fold, whereas dm5 had minimal effects (Fig. C and D). Western blotting analyses revealed that dm4 and dm5 were expressed at similar levels (data not shown). Therefore, HDAC4 represses MEF2C-dependent transcription through two repression domains. This may explain why substitution of histidines 802 and 803 had minimal effects on the ability of HDAC4 to repress MEF2C-dependent transcription (Fig. C). Surprisingly, the same mutation also had minimal effects on the ability of dm1 to repress MEF2C-dependent transcription, implying the existence of additional repression mechanisms. Taken together, these results suggest that through a MEF2C interaction domain and at least two repression domains, HDAC4 counteracts MEF2C-dependent activation of the c-jun promoter.