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J Biol Chem, Vol. 274, Issue 40, 28528-28536, October 1, 1999


Identification of a Human Histone Acetyltransferase Related to Monocytic Leukemia Zinc Finger Protein*

Nathalie ChampagneDagger §, Nicholas R. BertosDagger §, Nadine PelletierDagger §, Audrey H. WangDagger , Marko VezmarDagger , Yan YangDagger , Henry H. Heng, and Xiang-Jiao YangDagger parallel

From the Dagger  Molecular Oncology Group, Department of Medicine, McGill University Health Center, Montréal, Québec H3A 1A1 and the  Biology Department, York University, North York, Ontario M3J 1P3, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

We describe here the identification and functional characterization of a novel human histone acetyltransferase, termed MORF (monocytic leukemia zinc finger protein-related factor). MORF is a 1781-residue protein displaying significant sequence similarity to MOZ (monocytic leukemia zinc finger protein). MORF is ubiquitously expressed in adult human tissues, and its gene is located at human chromosome band 10q22. MORF has intrinsic histone acetyltransferase activity. In addition to its histone acetyltransferase domain, MORF possesses a strong transcriptional repression domain at its N terminus and a highly potent activation domain at its C terminus. Therefore, MORF is a novel histone acetyltransferase that contains multiple functional domains and may be involved in both positive and negative regulation of transcription.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

In eukaryotes, DNA is packaged into chromatin, a highly organized DNA-protein complex that fulfills important functions not only as a structural element in preserving genetic information but also as an active player in controlling gene activity (1, 2). How chromatin structure is regulated by DNA-binding transcription factors remains a central issue in studies of eukaryotic gene regulation. One regulatory mechanism involves acetylation of epsilon -amino groups of specific lysine residues located at the flexible N termini of core histones (1-5). Although transcriptionally silent heterochromatin is usually hypoacetylated, transcriptionally active euchromatin is hyperacetylated (6-8). Mechanistically, histone acetylation affects nucleosome stability and/or internucleosomal interaction or interferes with the interaction of histone tails with other proteins (4, 5, 9-11).

Histone acetyltransferases and deacetylases are the enzymes responsible for governing dynamic levels of histone acetylation at various chromatin domains in vivo (12, 13). Histone deacetylases have been found to be associated with transcriptional repression (14, 15). On the other hand, histone acetyltransferase (HAT) 1 activity is intrinsic to several known transcriptional coactivators, including GCN5 (general control nonderepressible 5) (16, 17), PCAF (18), p300 (the 300-kDa cellular protein associated with adenoviral E1A) (19), CBP (19, 20), and others (reviewed in Refs. 5, 13, 15, and 21-23).

Aberrant histone acetylation may lead to tumorigenesis (24, 25). One piece of supporting evidence is that the CBP gene is frequently rearranged in cancers (26-29). Interestingly, one of the translocation partners involved is the MOZ (monocytic leukemia zinc finger protein) gene (26, 30). MOZ itself contains a putative acetyl-CoA binding motif and shares its putative HAT domain with the yeast proteins SAS (something about silencing) 2, SAS3, and ESA1 (essential SAS2-related acetyltransferase 1) (31, 32), Drosophila MOF (males absent on the first) (33), and human TIP60 (HIV Tat-interacting protein of 60 kDa) (34). Among these proteins, ESA1 and TIP60 have been shown to possess intrinsic HAT activity (32, 34), whereas recombinant MOF does not exhibit any detectable HAT activity (33). Intriguingly, the putative yeast HATs, SAS2 and SAS3, have been implicated in both positive and negative regulation of gene expression (31, 35), and ESA1 has recently been found to be required for cell cycle progression (36). Furthermore, MOF, which is required for dosage compensation in male flies, has been shown to be targeted to the X chromosome of Drosophila (33, 37). For human MOZ, no biochemical or functional studies have been documented.

In this paper, we report the identification of a new human HAT, termed MORF (for MOZ-related factor) and further show that MORF possesses functional domains characteristic of a transcriptional regulator. Our results suggest the direct involvement of MORF and its related protein MOZ in transcriptional regulation and thus provide new insights into how abnormal forms of MOZ lead to tumorigenesis.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Molecular Cloning-- cDNA library screening, plasmid construction, and DNA sequencing were performed following standard procedures. Northern analyses on poly(A) RNA blots (CLONTECH) were carried out according to the manufacturer's instructions. The reporter tk-Luc was derived from pGL2 (Promega) by insertion of the thymidine kinase (tk) core promoter (-105/+52). Gal4-tk-Luc was constructed from tk-Luc by insertion of five copies of the Gal4-binding site upstream from the tk promoter. Gal4-E4-Luc was derived from Gal4-tk-Luc by replacement of the tk region with the adenoviral E4 core promoter from 3TP-Lux (38).

Fluorescence in Situ Hybridization-- FISH was performed on human lymphocytes as described (39), using as the probe a 5.8-kilobase pair MORF cDNA fragment biotinylated with dATP using the BioNick labeling kit (Life Technologies, Inc.).

Protein Expression and Purification-- Full-length MORF was expressed in Sf9 cells as a fusion protein with the FLAG epitope tag (IBI-Kodak) using a recombinant baculovirus generated with the Bac-To-Bac baculovirus system (Life Technologies, Inc.). The expressed fusion protein, f-MORF, was affinity-purified on M2-agarose and eluted with FLAG peptide (0.1 mg/ml; IBI-Kodak). The FLAG-tagged PCAF protein, f-PCAF, was similarly produced in and purified from Sf9 cells using the PCAF recombinant baculovirus previously described (18). Maltose-binding protein (MBP)-MORF mutants were expressed in Escherichia coli, purified on amylose resin (New England Biolabs), eluted with maltose (10 mM), and used directly for further analyses. For all affinity purification, buffer B (20 mM Tris-HCl, pH 8.0, 10% glycerol, 5 mM MgCl2, 0.1% Nonidet P-40, and protease inhibitors) containing 0.5 M KCl was used as lysis and washing buffers. In all elution buffers, the concentration of KCl was reduced to 0.15 M.

Western Blot Analysis-- Affinity-purified f-MORF and f-PCAF proteins were electrotransferred to BioTrace nitrocellulose membranes (Gelman Sciences) and probed with M2 anti-FLAG antibody (IBI-Kodak). Blots were developed with Supersignal chemiluminescent substrate (Pierce).

HAT Assay-- HAT activity was determined by analyzing incorporation of 3H- or 14C-labeled acetyl groups into histones. To measure HAT activity, Whatman P81 filter binding assays were used (40, 41). A typical reaction (20 µl) contained 75 nCi of [3H]acetyl-CoA (4.7 Ci/mmol; Amersham Pharmacia Biotech) and 2 µg of calf thymus histones (type IIa; Sigma). The reaction mixture was incubated at 30 °C for 10 min and then processed as described (40, 41). For acetyllysine peptides (19), 30 µg were used per reaction. To distinguish which histones were acetylated, each reaction (20 µl) contained 2.5 nCi of [14C]acetyl-CoA (51 mCi/mmol; Amersham Pharmacia Biotech) and 0.5 µg of HeLa octamers, nucleosomes, or oligonucleosomes. The reaction was carried out at 30 °C for 30 min and stopped by the addition of 10 µl of 3× SDS sample buffer, followed by separation on 15% SDS-PAGE gels and subsequent fluorography or phosphoimaging analysis using a FUJIX BAS 100 phosphoimager (18). Although the latter assay distinguishes which histones are acetylated, the P81 filter binding assay is more reliable and convenient for quantitative determination of HAT activity.

Reporter Gene Assays-- SuperFect transfection reagent (Qiagen) was used to transiently transfect a luciferase reporter (200 ng) and/or mammalian expression plasmids (200 ng) into NIH3T3 or 293T cells. pBluescript KSII(+) was used to normalize the total amount of plasmids used in each transfection, and a beta -galactosidase reporter driven by the cytomegalovirus promoter (50 ng) was cotransfected for normalization of transfection efficiency. After 48 h, luciferase activity of transfected cells was determined using D-(-)-Luciferin (Roche Molecular Biochemicals) as the substrate. Galactosidase activity was measured using Galacto-Light PlusTM (Tropix, Perkin-Elmer Co.) as the substrate. The chemiluminescence from activated Luciferin or Galacto-Light PlusTM was measured on a Luminometer Plate Reader (Dynex). Each transfection was performed at least three times to ensure that consistent results were obtained.

To verify the expression of Gal4 fusion proteins, expression plasmids were transfected to 293T cells; total or nuclear extracts were prepared for Western blotting analysis using a monoclonal anti-Gal4 antibody (Santa Cruz Biotech., RK5C1). Total extracts were prepared as described above using buffer B containing 0.15 M KCl. For nuclear extracts, transfected cells were washed twice with phosphate-buffered saline and lysed in situ using 5 ml (for a 10 cm dish) of ice-cold hypotonic lysis buffer (20 mM HEPES, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 25 mM NaF, 25 mM beta -glycerophosphate, 1 mM dithiothreitol, and protease inhibitors). After 5 min on ice, cell lysates were harvested by scraping and centrifuged for 5 min at 500 rpm on a Beckman swinging bucket tabletop centrifuge to pellet the nuclei. 0.1 ml of hypotonic lysis buffer containing 0.5 M NaCl was used to extract the nuclei. After being rotated for 20 min at 4 °C and brief centrifugation, the supernatants were collected and used as nuclear extracts for Western blotting analysis.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Cloning of MORF-- With known and putative HATs as baits, we performed BLAST and PSI-BLAST searches (42) against various sequence data bases. During these searches, we found a partial human cDNA clone (GenBankTM accession number AB002381). This partial clone encodes a polypeptide displaying significant sequence similarity to MOZ. To obtain the complete coding sequence, we screened human cDNA libraries. Polymerase chain reaction and sequence analyses of a majority of positive cDNA clones indicated that the full-length MORF clone encodes a polypeptide consisting of 1781 residues (Fig. 1A). The remaining positive clones were found to encode the polypeptides MORFalpha and MORFbeta , with 109 and 292 residues inserted between Pro372 and Asp373 of MORF, respectively (Fig. 1B). Data base searches and amino acid sequence comparison indicated that MORF is homologous to MOZ (identity, 60%; similarity, 66%). As shown in Fig. 1A, MORF is composed of four parts: an N-terminal region containing two C4HC3 PHD-zinc fingers, a putative HAT domain, an acidic region, and a C-terminal Ser/Met-rich domain. PHD-zinc fingers are putative protein-protein interaction motifs found in numerous proteins implicated in gene regulation, including the transcriptional regulator ATRX (43, 44), the corepressor KAP-1/TIF1-beta (45, 46), and the helicase protein Mi2 (47-51). Interestingly, the latter two are known transcriptional repressors. The putative HAT domain of MORF is homologous to that shared by MOZ (26), HBO1 (GenBankTM accession number AF074606), TIP60 (34), MOF (52), SAS2 (31), SAS3 (31), and ESA1 (32). The acidic and Ser/Met-rich domains of MORF do not display obvious sequence similarity to known proteins other than MOZ. These structural features of MORF suggest that it may be a HAT with novel properties.


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  		Fig. 1.   Primary structure of MORF and its isoforms. A, comparison of amino acid sequences of MORF and MOZ (GenBankTM accession number U47742). The sequences were aligned using the Bestfit program (Genetics Computer Group, Inc.). Putative domains are marked by dark lines at right. Cys and His residues coordinating zinc binding in the PHD- and C2HC-zinc fingers are shown in bold. Two putative nuclear localization signals (NLS1 and NLS2) and a putative acetyl-CoA-binding site are also indicated. A vertical arrow denotes the insertion site of extra residues in alternatively spliced variants. B, sequences of extra amino acids of MORFalpha (top) and MORFbeta (bottom) inserted between Pro372 and Asp373 of MORF.

As shown in Fig. 2A, Northern blot analyses of poly(A) RNA from various human tissues indicated that MORF is ubiquitously expressed, most abundantly in heart, pancreas, testis, and ovary. The expression of MORF is low in lung but detectable. FISH analyses revealed that the MORF gene is located at human chromosome band 10q22.2 (Fig. 2, B and C). A juvenile polyposis tumor suppressor locus has been mapped to 10q22 (53). Furthermore, this band is abnormal in a patient with biphenotypic acute leukemia (54), and amplification of a candidate gene located at 10q22.1-q23.1 has been correlated with the metastasis of bladder cancers (55). Therefore, the MORF gene is located at a chromosomal region that is rearranged in several neoplasms.


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  		Fig. 2.   Analysis of the MORF gene. A, expression of MORF among different adult human tissues. Poly(A) RNA blots were probed with an MORF cDNA fragment corresponding to its 3'-untranslated region. Relative positions of RNA markers are indicated at right. Each lane contains 2 µg of poly(A) RNA; hybridization with an actin cDNA probe confirmed that similar amounts of RNA are present in all lanes (data not shown). B, ideogram illustrating the chromosomal localization of MORF. Human blood lymphocytes were used for FISH. The hybridization efficiency was 86%, i.e. 86 of 100 mitotic figures checked showed this localization. Each dot represents double FISH signals (C) detected. C, example of FISH mapping. The left panel shows FISH signals on chromosome 10 (indicated by an arrow), whereas the right panel shows the same mitotic figure stained with 4',6-diamidino-2-phenylindole to identify chromosomes.

HAT Activity of MORF-- Next we asked whether MORF is really a HAT. To test this, we tried to express full-length MORF as a FLAG-tagged fusion protein in Sf9 insect cells. Due to unknown reasons, the recombinant baculovirus was not stable, and the expression level of f-MORF was very low. Because of the limited amount of MORF available, Western analysis with an anti-FLAG antibody was used to determine the concentration of FLAG-tagged MORF (f-MORF) in affinity-purified preparations (Fig. 3A, lane 2). For such Western analyses, FLAG-tagged PCAF (Fig. 3A, f-PCAF, lane 1), which could be highly expressed with a similar system and affinity-purified to near homogeneity (18), was utilized for comparison. To determine HAT activity of full-length MORF, both f-MORF and f-PCAF were affinity-purified and subjected to HAT assays. As shown in Fig. 3B, affinity-purified f-MORF was much more active than f-PCAF.


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  		Fig. 3.   HAT activity of full-length MORF. A, Western analysis of f-MORF. The FLAG-tagged MORF protein, f-MORF, was expressed in Sf9 cells and affinity-purified on M2-agarose. Western analysis of f-MORF with an anti-FLAG antibody is shown here (lane 2). For comparison, f-PCAF was similarly prepared and analyzed (lane 1). B, HAT activity of f-PCAF and f-MORF determined by P81 filter binding assays. Both f-MORF and f-PCAF were affinity-purified on M2-agarose from infected Sf9 insect cell extracts. During affinity purification, a buffer containing 0.5 M KCl was used for extensive washing; under such conditions, with uninfected Sf9 cell extracts, equivalent amounts of M2-agarose retained minimal HAT activity (data not shown). The amount (in ng) of f-PCAF and f-MORF used in the assays are indicated at the bottom. The amount of f-MORF was estimated based on Western analyses similar to that shown in (A) except that various amounts of f-PCAF were used to ensure that signals from such analyses were proportional to the molar amounts of FLAG-tagged proteins tested. C, substrate specificity of f-MORF. 0.5 µg of HeLa core histones (lanes 1-3) or oligonucleosomes (lanes 4-6) was incubated with no enzyme (lanes 1 and 4), f-PCAF (2.4 pmol; lanes 2 and 5), or f-MORF (0.06 pmol; lanes 3 and 6) in the presence of [14C]acetyl-CoA. After separation on SDS-PAGE gels, 14C-labeled proteins were detected by phosphoimaging.

To determine the substrate specificity, HeLa histones and oligonucleosomes were labeled with f-MORF in the presence of [14C]acetyl-CoA, resolved by SDS-PAGE, and subjected to fluorography or phosphoimaging analysis. As shown in Fig. 3C, f-MORF preferentially acetylated histones H3 and H4, whereas f-PCAF preferentially acetylated H3. Like f-PCAF, f-MORF was autoacetylated. When oligonucleosomes were used as substrates, f-MORF preferentially acetylated H4, whereas f-PCAF preferentially acetylated H3. Taken together, these results indicate that MORF is a potent HAT.

Characterization of the HAT Domain of MORF-- To map the HAT domain, we expressed several MORF fragments in E. coli as a protein fused to the following affinity tags: His6, glutathione S-transferase, and MBP. Among these, only the MBP fusion proteins could be expressed in soluble forms (Fig. 4A). These fusion proteins were affinity-purified on amylose resin (Fig. 4B). HAT assays were performed with these purified MBP fusion proteins. As shown in Fig. 4C, MBP-A efficiently acetylated histones. Deletion of residues 361-425 increased HAT activity by 4-fold (Fig. 4C, compare MBP-A and MBP-B), suggesting that residues 361-425 negatively regulate the activity of the HAT domain. On a molar basis, MBP-B was found to be about 40-fold less active than f-MORF. Further deletion of residues 426-460 abolished the activity (Fig. 4C, MBP-C). Deletion of residues 554-587, which contains the putative acetyl-CoA-binding motif, inactivated the enzyme (Fig. 4C, MBP-D). Altogether, these results indicate that residues 426-716 of MORF constitute its HAT domain. These results also support that MORF has intrinsic HAT activity.


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  		Fig. 4.   Characterization of the HAT domain of MORF. A, schematic representation of MBP-MORF fragments used to determine intrinsic HAT activity of MORF. The putative acetyl-CoA-binding site is indicated by the tripeptide motif GYG. B, analysis of affinity-purified MBP-MORF fragments. MBP-A, -B, -C, -D, and -E (0.4 µg/lane) were resolved on 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. C, HAT activity of MBP-MORF fragments determined by P81 filter binding assays. HAT activity is expressed in acetyl groups transferred (dpm) per pmol of MBP-fusion. D, substrate specificity of MBP-A and MBP-E. 0.5 µg of HeLa core histones was incubated with 2.5 pmol of MBP-A (lane 1) or MBP-E (lane 3) in the presence of [14C]acetyl-CoA. After separation on SDS-PAGE gels, 14C-labeled proteins were detected by phosphoimaging.

As shown in Fig. 1, MORFalpha contains an insertion of 109 residues between Pro372 and Asp373 of MORF. Because residues 361-425 of MORF serve as a negative regulator for the HAT activity, we tested whether the MORFalpha fragment corresponding to residues 426-716 of MORF has distinct HAT activity. For this, residues 361-825 of MORFalpha was expressed and purified as an MBP fusion protein. This fusion protein (Fig. 4C, MBP-E) was as active as MBP-A, suggesting that the insertion of 109 residues between Pro372 and Asp373 of MORF does not relieve the inhibitory effect of residues 361-425 on the HAT domain.

To determine which histones are acetylated, substrates labeled with MBP-A or MBP-E were separated by SDS-PAGE and subjected to fluorography or phosphoimaging analysis. As shown in Fig. 4D, MBP-A and MBP-E preferentially acetylated free histones H3 and H4. Unlike MBP-A, MBP-E was autoacetylated. When oligonucleosomes were used, no detectable acetylation was observed with either MBP-A or MBP-E (data not shown).

Next we sought to assess which lysine of histone H4 is acetylated. For this, histone H4 acetyllysine peptides (Fig. 5A and Ref. 19) were used as substrates. As shown in Fig. 5 (B---D), MBP-A, -B, and -E acetylated Lys5, Lys8, Lys12, and Lys16 of histone H4.


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  		Fig. 5.   Identification of lysine residues of histone H4 acetylated by MORF fragments. Chemically synthesized acetyllysine peptides (A) were incubated with MBP-A (B), -B (C), and -E (D), and incorporated [3H]acetyl groups were quantified by P81 filter binding assays. For each enzyme, the activity observed with the wild-type peptide was arbitrarily set to 100% to calculate the relative activity when acetyllysine peptides were used.

Consistent with the fact that MORF shares a conserved HAT domain with TIP60 and ESA1 (Fig. 1A and Refs. 32 and 34), these results indicate that the substrate specificity of MORF is similar to that reported for TIP60 and ESA1. With free core histones as a substrate, it has been reported that TIP60 and ESA1 acetylate H2A, H3, and H4 (32, 34). Furthermore, TIP60 acetylates Lys5, Lys8, Lys12, and Lys16 of histone H4 (56). However, unlike MORF, ESA1 and TIP60 were found to be unable to acetylate nucleosomal histones H2A, H3, and H4 (32, 34). Interestingly, unlike full-length MORF, MBP-A is unable to efficiently acetylate nucleosomal histones. In the case of TIP60, only the HAT domain was analyzed (34), so it is still unclear whether full-length TIP60 is able to acetylate nucleosomal histones. It is unclear whether this difference is due to different assay conditions employed in different studies.

The substrate specificity of MORF is clearly different from that of other HATs such as GCN5, PCAF, p300, and CBP (16, 18-20, 57, 58). One complicating factor is that most HATs exist as multisubunit complexes in vivo and recombinant catalytic subunits display properties (e.g. substrate specificity and specific activity) different from the corresponding complexes (59, 60). This may partially explain why f-MORF is more active than MBP-B because some Sf9 cellular proteins may tightly associate with f-MORF and affect its function. Relevant to this, recombinant Drosophila MOF was found to be inactive, although it is expected to be an active HAT in vivo (33). Another complicating factor is that properties of HATs are affected by the assay conditions employed (57). Therefore, further biochemical studies are needed to fully elucidate the properties of MORF in vivo and to compare them with those of other HATs.

Transcriptional Ability of MORF-- Because MORF has intrinsic HAT activity, we next examined whether MORF is able to regulate transcription when tethered to a promoter. For this, a series of constructs was engineered to express MORF or its deletion mutants fused to the Gal4 DNA-binding domain (Fig. 6A). As shown in Fig. 6B, full-length MORF and its mutant Delta CoA weakly repressed transcription (by 2- and 4-fold, respectively). In contrast, the deletion mutant N426 activated transcription by 5.9-fold. The weak effects of these fusion proteins on transcription may be due to their low protein expression levels, which were undetectable by Western blotting analysis with a monoclonal anti-Gal4 antibody (data not shown). These results suggest that there is an activation domain located at the C-terminal part of MORF and that the N-terminal region (residues 1-426) counteracts the function of this activation domain.


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  		Fig. 6.   Mapping of activation and repression domains of MORF. A, schematic representation of MORF and its deletion mutants. The name for each mutant is given at left, and residues that each mutant contains are also indicated. The mutant Delta CoA lacks residues around the putative acetyl-CoA-binding site (from 554 to 587). B, transcriptional activation by the C-terminal mutants of MORF. Mammalian vectors were constructed to express MORF or its deletion mutants fused to the C terminus of the Gal4 DNA-binding domain (residues 1-147) and cotransfected into NIH3T3 cells with the Gal4-tk-Luc reporter. Luciferase activities were normalized to internal beta -galactosidase controls and used to calculate relative activation potential (the activity without an effector was arbitrarily set to 1.0). C, transcriptional repression by the N-terminal mutants of MORF. Mammalian constructs expressing the indicated Gal4 fusion proteins were engineered as above and cotransfected into NIH3T3 cells with the reporter Gal4-E4-Luc or 3TP-Lux.

To map the activation domain, we tested several deletion mutants and found that one mutant (N1268) activated transcription by 333-fold (Fig. 6B). This mutant had minimal effects on tk-Luc, a reporter lacking Gal4-binding sites, suggesting that the observed effect on Gal4-tk-Luc is dependent on specific recruitment to the Gal4-binding sites. To further define the activation domain, two deletion mutants (N1564 and N1493) were constructed. Although N1564 stimulated transcription by 46-fold, N1493 was inactive. Western analysis indicated that N1493 was well expressed as N1564 and N1493 (Fig. 7A). Therefore, a transcriptional activation domain is located at the Ser-rich region of MORF, and the Met-rich region is required for the optimal function of this activation domain.


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  		Fig. 7.   Expression of MORF Gal4 fusion proteins. Mammalian vectors expressing indicated Gal4 fusion proteins were transfected into 293T cells. Total extracts (A) or nuclear extracts (B) were prepared for Western analysis with an anti-Gal4 antibody. Western analysis of total extracts for the Gal4 fusion proteins C426, C352, C215, and C207 yielded various nonspecific bands, so nuclear extracts were used.

As shown above, the N-terminal region of MORF counteracts the function of its C-terminal activation domain. This could be due either to the N-terminal region binding to the C-terminal activation domain and then inhibiting its activating function or to the N-terminal region itself being a transcriptional repression domain. To test the latter possibility, a series of constructs containing truncations from the C terminus of MORF was engineered and tested in reporter assays (Fig. 6A). As shown in Fig. 6C, with the reporter Gal4-E4-Luc, C426 and C352 repressed transcription by 43- and 33-fold, respectively. On the other hand, the deletion mutants C215 and C207 down-regulated transcription to a lesser extent (by 5.9- and 3.7-fold, respectively). The expression level of C207 was lower than the others (Fig. 7B). These mutants had minimal effects on 3TP-Lux, which lacks Gal4-binding sites (38). Therefore, the observed repression on Gal4-E4-Luc is dependent on specific promoter tethering, suggesting that the N-terminal region of MORF constitutes an active repression domain.

We then decided to investigate possible repression mechanisms. PHD-zinc fingers of Mi2beta have been found to be required (but not sufficient) for direct interaction with the histone deacetylase HDAC1 (48), raising the question of whether the PHD-zinc finger-containing repression domain of MORF directly interacts with HDAC1. To test this, HDAC1 was synthesized in vitro in reticulocyte lysates and subjected to pull-down assays with MBP-MORF (1-426) immobilized on amylose resin. These assays revealed, however, that there was no detectable interaction between MORF (1-426) and HDAC1 (data not shown). To further substantiate this, equivalent amounts of MBP and MBP-MORF (1-426) were immobilized on amylose resin and incubated with 293T cellular extracts. Subsequently, the amylose resin was extensively washed, and bound proteins were eluted and subjected to histone deacetylase assays. These assays revealed that MBP-MORF (1-426) did not retain more histone deacetylase activity than MBP (data not shown), suggesting that MORF (1-426) does not interact with a histone deacetylase. Consistent with this, transcriptional repression mediated by MORF (1-426) could not be relieved by treatment with the histone deacetylase inhibitor trichostatin A (data not shown). Taken together, these results indicate that the repression mediated by MORF (1-426) operates through a mechanism other than recruitment of a histone deacetylase.

Conclusion-- The data presented here demonstrate that MORF is a new HAT containing multiple functional domains. In additional to its HAT domain, MORF possesses a potent transcriptional activation domain located at its C terminus. This reflects a theme already described for p300 and CBP, both of which contain two activation domains independent of their HAT domains (reviewed in Refs. 5, 13, 22, and 23). On the other hand, PCAF does not appear to have additional activation domains besides its HAT domain (18). Different from known HATs, MORF contains a strong transcriptional repression domain located at its N terminus. This repression domain contains two PHD-zinc fingers. Similar zinc fingers have been found in known transcriptional repressors, e.g. TIF1-beta /KAP-1 (45, 46, 61) and Mi2 (47-51). Unlike MORF, Mi2 interacts with HDAC1 through its PHD-zinc fingers (48, 50, 51). MORF shares its HAT domain with a family of proteins, including human MOZ (26) and TIP60 (34), Drosophila MOF (52), and yeast SAS2, SAS3, and ESA1 (32, 52). Interestingly, although MOF is considered to be involved in gene activation, SAS2 and SAS3 are involved in both positive and negative regulation of gene expression (31, 34, 52). Our findings on MORF add another level of complexity to the function of this new family of proteins.

In summary, we have identified a new human protein termed MORF. MORF is ubiquitously expressed, and its gene is located at chromosome band 10q22, a region rearranged in several neoplasms. MORF has intrinsic HAT activity. Unlike known HATs, MORF possesses a transcriptional repression domain at its N terminus and an activation domain at its C terminus. Based on these findings, we speculate that through their multiple functional domains, MORF and its homolog MOZ participate in both positive and negative regulation of gene expression in vivo. Therefore, this study also illuminates how abnormalities of the MOZ gene lead to leukemogenesis.

    ACKNOWLEDGEMENTS

We thank T. Nagase, Kazusa DNA Research Institute (Japan) for the cDNA clone AB002381; V. V. Ogryzko and Y. Nakatani for acetyllysine peptides; J. Côté for HeLa histones and oligonucleosomes; W. M. Yang and E. Seto for HDAC1 cDNA; and J. Massagué for the reporter 3TP-Lux.

    FOOTNOTES

* This work was supported by the National Cancer Institute of Canada with an operating grant from the Terry Fox Run and in part by funds from the Medical Research Council of Canada (to X. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF113514, AF119230, and AF119231.

§ These authors contributed equally to this work.

parallel Medical Research Council of Canada Scholar. To whom correspondence should be addressed: Molecular Oncology Group, Royal Victoria Hospital, Rm. H5-41, 687 Pine Ave., West Montréal, Québec H3A 1A1, Canada. E-mail: yangxj@lan1.molonc.mcgill.ca.

    ABBREVIATIONS

The abbreviations used are: HAT, histone acetyltransferase; CBP, CREB-binding protein; PCAF, p300/CBP-associated factor; PHD, plant homeodomain (also called leukemia-associated protein domain); MBP, maltose-binding protein; CoA, coenzyme A; Luc, luciferase; HDAC1, histone deacetylase 1; tk, thymidine kinase; FISH, fluorescence in situ hybridization; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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