From the Leukemia Research Fund Centre at the
Institute of Cancer Research, London SW3 6JB, United Kingdom and the
§ Department of Medicine, Mount Sinai Medical Centre,
New York, New York 10029
Received for publication, December 18, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Histone deacetylases (HDACs) perform an important
function in transcriptional regulation by modifying the core histones
of the nucleosome. We have now fully characterized a new member of the
Class II HDAC family, HDAC9. The enzyme contains a conserved deacetylase domain, represses reporter activity when recruited to a
promoter, and utilizes histones H3 and H4 as substrates in vitro and in vivo. HDAC9 is expressed in a
tissue-specific pattern that partially overlaps that of HDAC4. Within
the human hematopoietic system, expression of HDAC9 is biased toward
cells of monocytic and lymphoid lineages. The HDAC9 gene
encodes multiple protein isoforms, some of which display distinct
cellular localization patterns. For example, full-length HDAC9 is
localized in the nucleus, but the isoform lacking the region encoded by
exon 7 is in the cytoplasm. HDAC9 interacts and co-localizes in
vivo with a number of transcriptional repressors and
co-repressors, including TEL and N-CoR, whose functions have been
implicated in the pathogenesis of hematological malignancies. These
results suggest that HDAC9 plays a role in hematopoiesis; its
deregulated expression may be associated with some human cancers.
In eukaryotes, the ability to dynamically form and maintain
distinct functional domains of chromatin is fundamental to nuclear processes, including regulation of gene transcription (1). The basic
repeating unit of chromatin is the nucleosome, which is a complex
consisting of 1.75 superhelical turns of DNA wrapped around a core
histone octamer comprising two subunits each of H2A, H2B, H3, and H4
(2). The N-terminal tails of histones H3 and H4 protrude from the
nucleosome and interact with the negatively charged DNA phosphate
backbone when in their highly basic, unmodified state (3). These tails
contain specific amino acids that are targets for a variety of enzymes,
producing diverse modifications including acetylation, methylation, and
phosphorylation. Acetylation is thus far the most widely studied and
involves substitution of the It is therefore important in studies of gene expression to address the
mechanisms by which HDACs effect specific changes in chromatin
structure. Thus far, 11 histone deacetylases that share homology
through their deacetylase domains have been characterized, and they may
be broadly divided into two classes on the basis of homology to the
yeast HDACs RPD3 (Class I) and HDA1 (Class II) (6-9). Class I HDACs
are nuclear proteins that are generally small in size (40-55 kDa) and
ubiquitously expressed (10-12). In contrast, Class II HDACs are larger
(ranging from 100 to 130 kDa), and expression patterns tend to be
tissue-specific with the exception of HDAC10, which is more widely
expressed (8, 9, 13-20). Furthermore, HDACs 4, 5, and 7 may, in
response to calcium signaling effected by
Ca2+/calmodulin-dependent kinases, be exported
from the nucleus to the cytoplasm upon binding 14-3-3 signaling adaptor
proteins (21-25). The function of both Class I and II HDACs is, in
part, mediated through association with transcriptional co-repressors
such as SMRT/N-CoR, and mSin3A (15, 26-31), and HDACs have also been found to interact directly with some transcription factors and repress
their target promoters (32, 33). At least with respect to HDAC3,
association with the co-repressor appears to play a role in the
regulation of its catalytic activity (34).
Here, we report the cloning and characterization of HDAC9, a member of
the Class II histone deacetylase family. HDAC9 contains 1069 amino
acids and functions as deacetylase both in vitro and in vivo, and this activity is essential for its associated
repression of gene expression. Furthermore, HDAC9 is alternatively
spliced to generate multiple protein isoforms that may harbor distinct biological activities and may be associated with human cancer. One of
these isoforms consists of the noncatalytic N-terminal region of HDAC9
(conserved in HDACs 4, 5, and 7) and has been previously identified in
Xenopus (MEF2-interacting transcriptional repressor (MITR))
(35) and human (histone deacetylase-related protein (HDRP)) (36,
37).
Isolation of HDAC9 cDNAs and Plasmid
Construction--
Full-length human HDAC9 was cloned from
Marathon-Ready Human Brain cDNA (Clontech)
using the sense primer 9F1 5'-ATGCACAGTATGATCAGCTCA-3' and the
antisense primer 9R1 5'-GTCACACACAGGAAATATCAG-3' (see Fig. 2 for the
cDNA sequence and translation). HDAC9
Double-stranded oligonucleotide primers were cloned into both ends of
the HDAC9 cDNA according to Ausubel et al. (38),
utilizing BclI (5' end) and BbsI (3' end) sites
to create a N-terminal FLAG-tagged cDNA containing
5'-BamHI-XhoI-3' ends. The cDNA was then
subcloned into the pSG5 vector (Stratagene) containing a modified
polylinker to create F-HDAC9. FLAG-tagged HDAC9
GST expression vectors were derived from pGEX-5X1 (Amersham
Biosciences) by subcloning indicated cDNAs in-frame with the coding region for glutathione S-transferase. Mammalian two-hybrid
expression vectors were derived from pM
(Clontech) by subcloning the indicated cDNAs
in-frame with the coding region for the GAL4 DNA-binding domain. The
GAL4uasx5-Tk-Luc reporter was derived from the pT109luc
(39) plasmid by inserting five copies of the GAL4 DNA-binding site
upstream of the minimal HSV-Tk promoter and luciferase (Luc) gene. The MEF2REx3-Tk-Luc reporter was also derived from the pT109luc
plasmid by inserting three copies of the consensus MEF2-binding site. The TELREx3-Tk-Luc reporter, as well as mammalian and
in vitro expression vectors for AML1, TEL, and TEL-AML1,
have been previously described (40). Mammalian and in vitro
expression vectors for BCL-6 (41), MEF2D (42), PLZF (43), full-length
and partial N-CoR (27, 44), HDAC1 (45), HDAC3 (10), HDAC4 (13), as well
as Sin3A and B (30), and SUMO-1 and -2 (46) proteins have been
described by others.
Cell Culture--
293T, COS-7 and tsCOS cells (47) were
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum (Sigma). Hematopoietic cell lines and the colon cancer cell line
SW-620 were maintained in RPMI 1640 medium with 10% fetal calf serum (Sigma).
Isolation of Cell Populations--
Adult peripheral blood was
taken with informed consent, and the low density cells (<1.077 g/ml)
were separated using Lymphoprep (Nycomed Pharma AS) and resuspended in
phosphate-buffered saline (PBS) containing 1% fetal calf serum
(Sigma). For sorting, the cells were stained separately with either
mouse monoclonal anti-CD19 (Dako) or fluorescein
isothiocyanate-conjugated anti-CD14 (Caltag-Medsystems) followed by
enrichment of positive cells using the standard MACS system protocol
(Miltenyi Biotec). Rat anti-mouse IgG1 beads were used to
isolate CD19+ve cells and anti-fluorescein isothiocyanate
beads for CD14+ve cells. Nonviable cells were excluded by
the addition of the dye To-pro-3 iodide (Molecular Probes) to presorted
cells. Enriched cells were analyzed and sorted to >99% purity using a
fluorescence-activated cell sorter (FACS Vantage SE; B.D.
Biosciences) at two wavelengths (using 530/30- and 660/20-nm band passes).
Histone Deacetylase Assays--
Immunoprecipitations for histone
deacetylase assays were essentially performed as described (16). 293T
cells (5 × 107 cells) were transfected with PolyFect
(Qiagen). One 10-cm dish was used per four HDAC assays. After 24 h, the cells were harvested and lysed in low stringency lysis buffer
(50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40) in the presence of a protease
inhibitor mixture (Roche Molecular Biochemicals). To control for
expression of different constructs, concentrations of protein extracts
were normalized with a modified Lowry assay (Bio-Rad), and Western blot
analysis was performed with the ECL procedure (Amersham Biosciences) as
described (38). Extracts were precleared by incubation with protein
10% v/v protein A/G-Sepharose (Sigma) overnight at 4 °C. Precleared
lysates were immunoprecipitated by incubation with 10% v/v M2
anti-FLAG-agarose (Sigma) overnight at 4 °C. Immune complexes were
recovered by washing three times with low stringency lysis buffer,
twice with lysis buffer containing 0.5 M NaCl (high
stringency), and twice with HDAC buffer (10 mM Tris-HCl, pH
8.0, 10 mM NaCl, 10% glycerol). For inhibition studies, the immunoprecipitated complexes were preincubated with trichostatin A
(400 nM) in HDAC buffer for 30 min at 4 °C.
Peptides corresponding to the N-terminal sequences of histone H3
(ARTKQTARKSTGGKAPRKQLC) and H4 (SGRGKGGKGLGKGGAKRHRC) were [3H]acetylated according to the manufacturer's
instructions (Upstate Biotechnology, Inc.), with 20,000 cpm used as
substrate per reaction. The beads were resuspended in 200 µl of HDAC
buffer containing acetylated peptide and 1 mM
phenylmethylsulfonyl fluoride. Histone deacetylase activity was
determined after incubation for 4 h at 37 °C, according to the
peptide manufacturer's instructions.
Chromatin Immunoprecipitation Assay--
293T cells (5 × 107 cells) were transfected by CaPO4
(Profection, Promega) with 5 µg of GAL4uasx5-Tk-Luc
reporter plasmid plus 10 µg of expression vector containing heterologous fusions of the GAL4 DNA-binding domain with the indicated cDNAs. Immunoprecipitation of plasmid DNA plus associated histones was carried out ~40 h after transfection according to a previously published protocol (48), with the following modifications. Histone-DNA complexes were cross-linked by the addition of 1% formaldehyde to the
medium and incubation at 37 °C for 10 min. After lysis, the
chromatin was sonicated to 0.2-1.0 kb and diluted 10-fold in IP buffer
(0.01% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM
Tris-HCl, pH 8.0, 150 mM NaCl, plus protease inhibitors).
Protein samples for Western blotting were taken prior to dilution;
control samples for assaying input DNA were taken after dilution and
de-cross-linked. Anti-acetylated histone H4 polyclonal antibody
(Upstate Biotechnology, Inc.) was used for the immunoprecipitation, and
the DNA-histone complexes were collected overnight with protein
A/G-Sepharose beads (Santa Cruz). Sequences spanning the GAL4-binding
site in the reporter were detected by semi-quantitative PCR using the forward primer 5'-ATTGCAGCTTATAATGGTTA-3' and the reverse primer 5'-TTCGAATTCGCCAATGACAA-3'. The number of cycles was determined empirically to give results that fall within the linear range of this
particular PCR assay, and the reactions were visualized by agarose gel
electrophoresis followed by ethidium bromide staining.
RNA Isolation and Reverse Transcriptase-PCR Analysis--
Total
RNA was extracted from purified cells and cell lines using an
RNeasy kit (Qiagen). cDNA was made from total RNA by reverse transcription using Moloney murine leukemia virus (Invitrogen) or
Omniscript (Qiagen) reverse transcriptases in accordance with the
manufacturers' instructions. HDAC9 cDNA was primed with
5'-TAAAACAGAGGCAGCAGGGGAAGAGTGAGT-3' and HDAC9 Preparation of an HDAC9-specific Antiserum--
A rabbit
antiserum specific to the C terminus of human HDAC9 was raised using a
synthetic peptide corresponding to amino acids 1046-1060 of the
protein (DVEQPFAQEDSRTAG), which was conjugated to diptheria toxoid
(Mimotopes). This C-terminal region is not conserved in any other HDACs
closely related to the HDAC9 protein.
Antibodies to BCL-6 (D-9), GAL4 (DBD) (RK5C1), N-CoR
(C-20), TEL (C-20), TEL (N-19), HDAC4 (H-92), HDAC5 (P-16), HDAC6
(H-300), and HDRP/MITR (G-15) (this antibody also recognizes
full-length HDAC9 protein) were obtained from Santa Cruz Biotechnology.
Antibodies to Myc tag (9B11), HDAC1, HDAC3, and HDAC7 were obtained
from Cell Signaling Technology, and anti-FLAG M2 antibody was obtained from Sigma. Anti-mouse and anti-rabbit horseradish peroxidase-linked secondary antibodies used in chemiluminescent detection of Western blots were purchased from Amersham Biosciences.
GST Pull-down Assays--
All of the GST fusion proteins were
prepared using standard procedures (43).
[35S]Methionine-labeled proteins were synthesized
in vitro using a rabbit reticulocyte-coupled
transcription-translation system (Promega), following the supplier's
directions. 35S-Labeled proteins were incubated with 1 µg
of GST or a given fusion protein. The assays were performed in NETN
buffer (20 mmol/liter Tris, pH 8.0, 100 mmol/liter NaCl, 1 mmol/liter
EDTA, 0.5% Nonidet P-40) at 4 °C for 1 h with gentle rocking.
Glutathione-Sepharose beads were washed five times with H buffer (20 mmol/liter HEPES, pH 7.7, 50 mmol/liter KCl, 20% glycerol, 0.1%
Nonidet P-40). The bound proteins were eluted in Laemmli loading buffer
and separated by SDS-PAGE. The gels were fixed in 25% isopropanol and
10% acetic acid, dried, and exposed to Biomax film (Kodak).
Co-immunoprecipitation Assays--
10-cm dishes containing tsCOS
cells were transfected with 2 µg each of expression constructs
encoding N-CoR, TEL, and FLAG-tagged HDAC9 isoforms using PolyFect
(Qiagen). After 36 h the cells were washed in PBS and harvested,
and the immunocomplexes were isolated using the Catch and Release
immunoprecipitation system (Upstate Biotechnology, Inc.) according to
the manufacturer's instructions. Co-immunoprecipitated HDAC9 isoforms
were resolved by SDS-PAGE, blotted, and detected with anti-FLAG M2
antibody. Endogenously expressed proteins were co-immunoprecipitated as
above and detected with affinity-purified anti-HDAC9 rabbit polyclonal antibody.
Double Immunofluorescence Microscopy--
COS-7 cells growing on
coverslips were transfected using PolyFect (Qiagen). After 36 h,
the cells were fixed with 100% methanol at Confocal Immunofluorescence Microscopy--
104 REH
cells in log phase growth were cytospun and fixed in 4%
paraformaldehyde for 15 min at room temperature. The samples were
subsequently processed as described above. The cell nuclei were stained
with To-pro-3 iodide (Molecular Probes), and slides were mounted with
Vectashield (Vector Laboratories). Affinity-purified anti-HDAC9 rabbit
polyclonal antibody was used for detection, and imaging was performed
on a Leica TCS-SP2 system.
In Vivo Sumoylation Assays--
In vivo sumoylation
assays were carried out using the nickel affinity pull-down technique
(49). Briefly, tsCOS cells in 10-cm dishes were transfected with 2 µg
each of expression constructs encoding F-HDAC9, F-HDAC9 Cloning of Full-length HDAC9 cDNA--
The
GenBankTM (50) high throughput genomic sequence data base
(htgs) was searched with the amino acid sequences corresponding to the
deacetylase domains of various Class I and II HDACs. Several DNA
sequences encoding peptides with significant homology to HDAC5 were
found on a human BAC clone RP11-8I15 (accession number
AC016186), which contains 70 unordered contigs and maps to chromosome
18. When the GenBankTM nucleotide data base was searched
with a composite of the novel sequences showing homology to HDAC5, it
aligned exactly with BAC clone CTB-13P7 (accession number AC002088),
which mapped to chromosome 7p21.1, indicating that clone RP11-8I15 had
been submitted incorrectly to the htgs data base. A search of the
expressed sequence tag data base (dbEST) revealed that cDNA
corresponding to the putative histone deacetylase had been identified
in germinal center B cells (accession number AA287983); therefore
several hematopoietic cell lines were analyzed by RT-PCR with
oligonucleotide primers specific for the putative HDAC sequence and
also HDRP/MITR, which had been mapped to 7p15-p21 (35). In addition to
the amplifying the putative HDAC sequence itself; it was found that
this primer set also amplified cDNA from HDRP/MITR through to the
putative HDAC domain. The Ensembl Genome Server (51) indicated
an open reading frame (ORF) containing 22 exons (~2700 bp of
sequence), and RT-PCR was performed using oligonucleotide primers from
within the known sequence of the putative HDAC9 cDNA established by
us and the expected stop codon of the ORF, but no products were
observed. These data, together with the observed conservation of
C-terminal amino acid residues among other Class II HDACs indicated the
absence of the entire ORF. 3'-Rapid amplification of cDNA ends was
performed but failed to yield the remaining sequence, so
GenBankTM was searched for overlapping expressed sequence
tags to "walk" along the cDNA (Fig.
1B). This analysis revealed
that the sequence corresponding to the final five exons of the putative
HDAC gene contained in BAC clones RP5-1194E15 and GS1-465N13
(accession numbers AC004994 and AC004744, respectively) had been
submitted in the antisense direction in relation to the rest of the
gene (Fig. 1C). These clones accounted for almost 160 kb of
DNA, and subsequent analysis with them in the correct orientation
provided a genomic sequence that matched the ORF that we had generated from overlapping expressed sequence tags and, when translated, showed
significant homology with the amino acid sequences for HDACs 4, 5 and 7 (see Fig. 3A). We designated this novel gene HDAC9. An ORF mapping to the HDAC9 locus has been
independently cloned by another group (7); however, it does not encode
the full-length protein (see further below).
The HDAC9 Gene Is Differentially Spliced to Encode Multiple
Isoforms--
The full-length product of the HDAC9 gene comprises 1069 amino acids as shown in Fig. 2 and is
encoded by exons 2-26 (exon1 is untranslated). The 26 exons that form
the HDAC9 cDNA span ~500 kb of genomic sequence on 7p21.1. The
isoform that lacks the catalytic domain, HDAC9
Phylogenetic analysis of HDAC9 shows that it is a member of the Class
II histone deacetylases and is most closely related to HDAC5. The
recent discovery of HDACs 10 and 11 indicate that, based on analysis of
HDAC catalytic domain (Fig.
3B) and whole protein (Fig.
3C), there is a subdivision of the Class II histone deacetylase group consisting of HDACs 6, 10, and 11.
HDAC9 Possesses Deacetylase Activity--
To determine whether
HDAC9 possesses histone deacetylase activity, an in vitro
assay was performed using anti-FLAG immunoprecipitated HDAC9. As shown
in Fig. 4A, HDAC9 catalyzes
the deacetylation of peptides corresponding to the N-terminal tails of
both histone H3 and H4 with overall activity comparable with that of
HDAC4. Additionally, the Class II HDACs 4 and 9 appear to deacetylate the histone H4 peptide substrate less effectively when compared with
HDAC1. The value for deacetylation of histone H4 peptide is 44% for
HDAC4 and 36% for HDAC9, respectively, of the value observed for
histone H3 peptide. This contrasts with HDAC1, which deacetylated
histone H4 peptide with 70% of the activity seen for histone H3
peptide. These data are specific for a given HDAC, because a high
stringency wash removes endogenous Class I and II HDACs present in the
lysate that are not directly bound by anti-FLAG M2 agarose beads (Fig.
4B). Moreover, isoforms of HDAC9 that either lack
(HDAC9 Distribution of HDAC9 mRNA Is Tissue-specific and May Be
Deregulated in Human Cancers--
Analysis of HDAC4, HDAC9, and
HDAC9
When HDAC9 and HDAC9
Samples from colon cancer cell lines showed increased expression of
HDAC9 relative to normal colon tissue HDAC9 mRNA (Fig. 5C, lanes 31-34), especially variants lacking
exons 7 and 12 in SW-620. In general, there is greater variety of
expression of relative amounts of differentially spliced HDAC9
transcripts in samples containing the catalytic domain of HDAC9 than
those lacking it. One exception is bone marrow (Fig. 5C,
lane 2), where there is very little expression of HDAC9 HDAC9 Isoforms Interact Differentially with Co-repressors and
Proteins Implicated in Hematalogical Malignancies--
Previously, we
and others have demonstrated involvement of histone deacetylases in the
pathogenesis of both lymphoid and myeloid neoplasms (40, 56, 57). Given
the expression of HDAC9 in cell lines and samples derived from B cell
tumors, we examined whether it could interact with any oncogene
products known to recruit HDAC-containing complexes and to be involved
in B cell malignances. As anticipated, HDAC9 was found to associate
in vitro with the TEL protein, which is fused to AML1 in
pre-B cell childhood acute lymphoblastic leukemia (58, 59), and also
BCL-6, which is frequently associated with B cell neoplasias (60) (Fig.
6A, lanes 3 and
4). HDAC9 also interacted with HDACs 3 and 4 (Fig. 6A, lanes 1 and 2) and HDAC1 and PLZF
(data not shown). In addition, HDAC9 was found to interact with the
co-repressors mSin3A, mSin3B, and N-CoR, whose activities have been
implicated in the mechanism of action of several human cancers (61)
(Fig. 6A, lanes 5-8). These data indicate that
N-CoR contacts HDAC9 at multiple points, with the C-terminal region of
N-CoR also interacting with the catalytic domain of HDAC9.
Additionally, the catalytic domain of HDAC9 was found to associate with
mSin3A but not mSin3B.
The ability of HDAC9 expressed in vivo to interact with
N-CoR or TEL was also examined. Consistent with the in vitro
data, full-length HDAC9 isoforms were found to interact with N-CoR and TEL (Fig. 6B, lanes 12 and 17).
However, TEL co-precipitated poorly with HDAC9
The in vitro and in vivo interaction data between
HDAC9 and N-CoR and TEL are corroborated by specific patterns of
cellular localization between various isoforms of HDAC9 and its
interacting partners. Fig. 7A
shows the cellular localization of various isoforms of FLAG-tagged
HDAC9 when visualized either with anti-FLAG antibody or an antibody to
an N-terminal epitope of HDAC9. Note that HDAC9
To further analyze the interactions of HDAC9 and its isoforms in the
context of the cell, we performed double immunofluorescence assays.
When HDAC9 was co-expressed with N-CoR (Fig. 7C), they co-localize within the nucleus, and N-CoR assumes a more diffuse nuclear distribution that is identical to that seen with HDAC9 alone.
However, when N-CoR was co-expressed with HDAC9 HDAC9 Represses Transcriptional Activity in Vivo--
It has been
previously established that HDACs repress transcription when tethered
to DNA as Gal4 fusion proteins (11). As expected from earlier
experiments (Fig. 4, A and C), this effect is
also observed with HDAC9, HDAC9
The ability of HDAC9 to associate with transcriptional repressors, such
as TEL for example, suggested that HDAC9 could play a role in
repression of promoter activities by these proteins. To evaluate this
for the TEL protein, HDAC9 and just the HDAC9 catalytic domain were
co-transfected with TEL expression vector and a luciferase reporter
gene containing three copies of the TEL consensus binding site
(5'-TAAACAGGAAGT-3'). As expected, the addition of HDAC9 potentiated
repression by TEL, and the degree of repression observed was greater
for TEL plus full-length HDAC9 than for TEL plus HDAC9
When Sparrow et al. (35) first identified the
Xenopus homologue of HDAC9 Deletion of HDAC9 Exon 12 Results in the Loss of a Site Modified by
Sumoylation--
To test whether the different HDAC9 isoforms
containing the catalytic domain are endogenously expressed protein,
Western blot analysis was performed on whole cell lysates from tsCOS
cells transfected with different isoforms of HDAC9 (Fig.
9, lanes 1-3) and selected
hematopoietic and colon cancer cell lines that were used in RT-PCR
analysis, plus the T-cell leukemia cell line JM-1 (Fig. 9, lanes
4-9). The anti-HDAC9 rabbit polyclonal antibody used specifically
recognizes endogenous proteins expressed in cells positive for HDAC9
mRNA isoforms (Fig. 5C). Although overall protein and
mRNA levels are comparable between the different samples examined,
the full-length protein isoform is the most abundant species. This
could be due to decreased efficiency of translation and/or protein
isoform stability. The Western analysis shows that HDAC9 has an
apparent molecular mass of around 160 kDa, which is greater than
the predicted molecular mass of 117.5 kDa and indicates that HDAC9 has
undergone post-translational modifications. Interestingly, the
HDAC9 In this manuscript, we describe the full and complete cloning and
characterization of the ninth member of the histone deacetylase family,
HDAC9. The HDAC9 gene is located at 7p21.1, a region
implicated in neurological disorders (66-69) and a variety of cancers
including colorectal cancer (70), fibrosarcoma (71), childhood acute lymphoblastic leukemia (72), Wilms' tumor (65), and peripheral nerve
sheath tumors (73). The 3' end of the HDAC9 ORF is located only approximately 150 kb from the TWIST gene, which is
implicated craniosynostosis-associated Saethre-Chotzen syndrome
(74-76). Investigation of the genomic neighborhood of the
TWIST gene showed that deletions of DNA that encompassed the
HDAC9 locus led to a more severe phenotype with significant
learning difficulties (68). The potential involvement of HDAC9 in
central nervous system development and the pathogenesis of
Saethre-Chotzen syndrome is corroborated by its expression in the
developing brain.
The possibility that gene dosage effects involving HDAC9 are important
is also underlined by the correlation of increased HDAC9 expression in
colon cancer cell lines with the finding that around 37% of colon
cancers possessed gains of DNA sequence corresponding to 7p21 (70). The
HDAC9 gene comprises 26 exons and spans ~500 kb (more than
12 times the size of HDAC5 (77) and almost 40 times that of
HDAC3 (78)), and this may render it more susceptible to the
effects of genomic instability. Comparison of the mouse open reading
frame with human HDAC9 shows a high degree of conservation (data not
shown), including the potential for the same in-frame alternative
splicing. This point is reinforced when a comparison is made of the
noncoding genomic DNA contained within the mouse and human HDAC9 genes.
There are zones of high homology present, especially in the regions
adjacent to the regulated exons 7 and 12.
A sequence representing an incomplete open reading frame of full-length
HDAC9 has been recently reported by Zhou et al. (7). It is
possible that this sequence is a truncated isoform of HDAC9 lacking
exons 25 and 26. Because sequences encoded by exons 25 and 26 are
largely conserved among Class II HDACs and comprise the very C terminus
of the catalytic domain, it is not surprising that the truncated HDAC9
product reported by Zhou et al. (7) possessed very little
deacetylase activity (only 10% of the activity of HDAC4). This clearly
contrasts with the activity level of the full-length HDAC9 protein
reported here, which is close to that of HDAC4 (Fig.
4A).
Until recently, expression analyses indicated that both Class I and II
HDACs existed as single isoforms (11-13, 16). This is no longer the
case with respect to Class II HDACs because major variants have been
detected for HDAC7 and 10 (8, 15, 18-20). It should be noted that Zhou
et al. (7) also reported the presence of an alternate
isoform of HDAC9 that consisted of a transcript lacking exons 21-26
and encoding 19 residues of unique sequence, in much the same way as
HDAC9 The other differentially spliced isoform investigated was
HDAC9 In summary, the HDAC9 gene encodes multiple, functionally
distinct, and differentially expressed protein isoforms. Gene
regulation arising from alternative splicing is recognized as an
increasingly important factor in the creation of proteome diversity
(82, 83). The genomic size and degree to which HDAC9 is regulated is
unprecedented among other HDACs, and it perhaps reflects the complexity
of a system controlling its activities and may indicate a wider role
for its function than just histone and/or protein modification.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of specific lysines in
a process catalyzed by histone acetyltransferases. This leads to
a more acidic residue and an overall decreased affinity for DNA by the
histone octamer. For the transcriptional machinery, the packaging of
DNA into nucleosomal arrays presents a major physical obstacle in
gaining access to the DNA template, and there has long been evidence
that unwinding of nucleosomes because of the acetylation of histone
tails plays an important role in the activation of transcription (4).
As expected, enzymes that remove these modifications, histone
deacetylases (HDACs),1 are
important in gene silencing, and recent studies have implicated abnormal HDAC function in a number of human cancers (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD/MITR/HDRP was cloned
from Marathon-Ready Human Brain cDNA using the sense primer 9F1 and
the antisense primer 9
CDR1 5'-TCAGATAATGACTTTAATTACAAAT-3'. HDAC9
exon7 and HDAC9
exon12 were cloned from the acute monocytic leukemia cell line MONO-MAC-6 using the sense primer 9F1 and the antisense primer 9R3 5'-TCTCTAATCCATCCATGCCAA-3'. HDAC9
exon15 was
cloned from the acute Pre-B ALL cell line REH using the sense primer
9F2 5'-AGGCTGCTTTTATGCAACAG-3' and the antisense primer 9R2
5'-CTGAATGCTTCAAGGTACTCA-3'. See Fig. 1A for a schematic
detailing the positions of the above primers. PCR products were cloned
into pCRII (Invitrogen) and sequenced using BigDye (Perkin Elmer).
CD (F-HDAC9
CD) was
created by subcloning a 5'-PstI-XhoI-3' 3'
fragment from HDAC9
CD into F-HDAC9. Plasmids containing the various
FLAG-tagged alternatively spliced variants of HDAC9 and HDAC9
CD were
constructed by subcloning appropriate fragments from the partial
cDNAs described above into F-HDAC9 and F-HDAC9
CD.
CD/MITR cDNA was
primed with 5'-TCAGATAATGACTTTAATTACAAATCCTGG-3'. Glyceraldehyde-3-phosphate dehydrogenase cDNA was primed with random hexamers (Roche Diagnostics). 2 µg of total RNA was used per
20-µl reaction, and 2 µl of cDNA was used per subsequent PCR. The primer pairs were used for semi-quantitative PCR are shown in Fig.
5A. PCRs were visualized by agarose gel electrophoresis and
ethidium bromide staining.
20 °C for 10 min and
washed three times for 5 min in PBS. The cells were permeabilized with
0.5% Triton X-100 in PBS and washed three times for 5 min in PBS. The
cells were blocked for 30 min at room temperature in 2% bovine serum
albumin (Vector Laboratories) and 5% relevant serum (Jackson
Immunoresearch Laboratories, Ltd.) for the secondary antibody to be
used. The primary antibodies were diluted to the appropriate
concentrations in blocking buffer, centrifuged at 4 °C for 20 min,
and then incubated at 4 °C overnight. The cells were washed three
times for 15 min in PBS and incubated with the appropriate fluorescein
isothiocyanate- or TRITC-conjugated secondary antibody (Jackson
Immunoresearch Laboratories, Ltd.) for 2 h at room temperature.
The cells were washed three times for 15 min in PBS and mounted on
slides using Vectashield with DAPI (Vector Laboratories).
Imaging was performed on a Zeiss Axioplan2 microscope with a cooled CCD
camera (Photometrics Quantix) using Smartcapture 2 software (Digital Scientific).
exon12, and
polyhistidine-tagged SUMO-1 or -2 using PolyFect (Qiagen). After
36 h the cells were washed in PBS and harvested directly in 1 ml
of guanidine lysis buffer (6 M guanidine HCl, 100 mM NaCl, 10 mM Tris, 50 mM
NaH2PO4, pH 8.0), sonicated, and centrifuged.
The cleared samples were incubated for 2 h with 20 µl (packed
volume) of Talon nickel affinity beads (Clontech).
The bound proteins were washed twice in lysis buffer, three times in
urea buffer (8 M urea, 100 mM NaCl, 50 mM NaH2PO4, pH 6.5), and once in
cold PBS before being eluted by boiling in Laemmli loading buffer. 20%
of each guanidine lysis sample was removed and precipitated for 15 min
on ice with 5% trichloroacetic acid. Samples were centrifuged, washed
in 100% ethanol, and resuspended in Laemmli loading buffer. The
proteins were separated by SDS-PAGE and detected with affinity-purified
anti-HDAC9 rabbit polyclonal antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Isolation of HDAC9 cDNA and analysis of
the HDAC9 gene. A, schematic representation
of the two major HDAC9 isoforms together with the oligonucleotide
primers (indicated as arrows) used in cloning of their
respective cDNAs. B, identification of the 3' sequence
of the HDAC9 open reading frame. The shaded letters
represent the open reading frame derived by using overlapping expressed
sequence tags (indicated by GenBankTM accession number) to
walk along chromosome 7p21.1. C, genomic organization of
HDAC9. The accession numbers of the clones that comprise the HDAC9 gene
are shown together with the exon positions on chromosome 7, which were
established using the BLAT alignment tool (84). HDAC9
exon/intron splice junctions are also detailed with consensus splice
donor and acceptor sequences between exons (uppercase
letters) and introns (lowercase letters)
underlined. Exons highlighted in gray have the
potential to be spliced out in-frame. exon7 and
exon12 cDNAs
have been detected by RT-PCR.
CD (HDRP/MITR), is 593 amino acids in length and contains 16 residues of unique sequence
encoded by a region of exon 12, which is 3' to the splice donor site
used to generate the HDAC9 ORF (Fig. 2). There are several exon
deletions that may occur that naturally preserve the open reading frame
of HDAC9 (Fig. 1C), and two have been identified and cloned.
It has been previously suggested that HDAC9 is potentially
alternatively spliced at exon7 (7), and this is indeed the case.
HDAC9
exon7 is 1025 amino acids long and contains an Ala
Glu
substitution at position 222 as the result of the deletion of exon 7. This isoform lacks two serines (Ser223 and
Ser253), which when phosphorylated have been implicated in
14-3-3 protein-dependent shuttling of HDAC4 and 5 from the
nucleus to the cytoplasm, and also a tripartite nuclear localization
signal (21, 23, 52). Exon 12 may also be deleted in-frame to generate
an alternate protein isoform that is 981 amino acids long. The region
encoded by exon 12 contains a conserved sumoylation site identified in HDAC4 (53) and a potential leucine zipper motif that may mediate interactions between HDAC9 and other proteins. cDNA that encodes an
isoform possessing neither exon 7 nor exon 12 has also been cloned.
HDAC9
exon15 is 1027 amino acids long and lacks a region within the
catalytic domain adjacent to the active site. This isoform does not
naturally conserve the ORF of HDAC9 and may have undergone RNA
editing.
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Fig. 2.
HDAC9 cDNA and predicted amino acid
sequence. HDAC9 full-length cDNA has been cloned using human
brain mRNA. It contains an open reading frame of 3210 bp, which
yields a 1069-amino acid protein. The amino acids are indicated in
single-letter code and are numbered on the right. The
positions of 25 introns and their sizes in bp are also indicated. The
HDAC9 exon7 isoform results in an Ala
Glu substitution at
position 222. The sequence specific to HDAC9
CD exon 12 is shown in
italics.
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Fig. 3.
Phylogenetic analysis of HDAC9. A,
amino acid sequence alignment of HDAC9, HDAC4, HDAC5, HDAC7, and a
bacterial deacetylase, HDLP. The indicated sequences were aligned using
Clustal W. Identical residues are boxed and highlighted in
dark gray; similar residues are shaded in
light gray. B, evaluation of amino acid
identities and similarities of HDAC9 deacetylase domain compared with
those of HDAC1 and other Class II histone deacetylases. The histone
deacetylase domains are shown in light gray. The percentage
values were obtained by comparing the deacetylase domain of HDAC9 with
the indicated protein sequences on the BioEdit Sequence Alignment
Editor using the Blosum62 matrix. C, phylogenetic tree of
HDAC1 through to HDAC11. The sequences were aligned using the Clustal W
server at the Center for Molecular and Biomolecular Informatics
(University of Nijmegen, Nijmegen, Holland). The PHYLIP notation
output was used to construct an unrooted tree with Unrooted (Manolo
Gouy, University Claude Bernard, Lyon, France).
CD) or possess a deletion (HDAC9
exon15) in the catalytic
domain do not deacetylate histone H3 peptide effectively (Fig.
4A). This indicates that co-immunoprecipitation of
uncharacterized proteins possessing histone deacetylase activities or
known HDACs at levels not detected by our analysis is not likely to
account for the observed in vitro activity of HDAC9. The
in vitro assay results are corroborated in vivo
by chromatin immunoprecipitation analysis (Fig. 4C). There
is a difference in the amount of target DNA sequence that may be
co-immunoprecipitated with acetylated histone H3, compared with
acetylated histone H4, when proteins containing a heterologous fusion
of GAL4 DNA-binding domain to the catalytic domain of HDAC9 or to a
lesser extent the whole HDAC9 protein are tested. This result is not
observed for HDAC9
CD and is most likely a reflection of the ability
of this N-terminal isoform of HDAC9 lacking the catalytic domain to
bind Class I and II HDACs in vivo (54).
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Fig. 4.
HDAC9 possesses histone deacetylase activity.
A, HDAC9 deacetylates histone H3 and H4 peptide in
vitro. 293T cells were transfected with FLAG-tagged HDACs as
indicated. Whole cell lysates were produced, and the HDACs were
precipitated with anti-FLAG agarose. The precipitates were thoroughly
washed and assayed for their ability to deacetylate
[3H]acetyl-labeled peptides corresponding to the N
terminus of histones H3 or H4. Free acetate was extracted, and the
number of cpm were measured by liquid scintillation. The HDAC inhibitor
trichostatin A was added to control reactions as indicated. The cells
were transfected with pSG5 empty vector (Stratagene) as a negative
control. This is taken to be the background level of deacetylase
activity measured (dark gray). B, under
high stringency conditions, HDAC9 does not co-immunoprecipitate with
other previously characterized members of the histone deacetylase
family. 293T cells were transfected with FLAG-tagged HDAC9 and
immunoprecipitated as described above. The precipitates were then
immunoblotted with antibodies raised against the indicated HDACs. Aside
from HDAC9, input values for the indicated HDACs show levels of the
endogenous enzyme. C, HDAC9 causes hypoacetylation of target
genes in vivo. 293T cells were transfected with
GAL4uasx5-Tk-Luc reporter together with GAL4 DNA-binding
domain fusions as indicated. GAL4DBD-HDAC9CD contains only the
catalytic domain of HDAC9. The GAL4 reporter is derived from the
pT109luc plasmid (39). This plasmid contains a SV40 origin of
replication, which has been shown to induce chromatinization of plasmid
DNA in cell lines expressing large T antigen (85). Soluble chromatin
preparations from the transfections were immunoprecipitated with
antibodies against acetylated histone H3 ( AcHistone H3) or H4
(
AcHistone H4) (Upstate Biotechnology, Inc.) and analyzed by
semi-quantitative PCR. As negative and positive controls, soluble
chromatin preparations were immunoprecipitated with IgG or anti-GAL4
(DBD) (RK5C1) antibody, respectively. Aliquots of the chromatin were
also analyzed before immunoprecipitation (DNA input). The
numerical values, which reflect relative abundance of acetylated
chromatin, were obtained by densitometric analysis using Labworks
analysis software (Ultra-violet Products).
CD expression in various human tissues and cell lines reveals
that not only are there differences in the pattern of expression
between HDAC4 and HDAC9 but also between the transcripts encoding the
two major isoforms of HDAC9 (Fig.
5B). For example, all three
transcripts were expressed in skeletal muscle and the adult and fetal
brain (Fig. 5B, lanes 3 and 12),
although HDAC9 and HDAC9
CD were considerably more abundant in fetal
tissue. HDAC4 alone is expressed in kidney, liver, and the myeloid
leukemic cell line HL60 (Fig. 5B, lanes 7,
8, and 16). Although expression of HDAC9 was very
low or absent in heart, T cell leukemia cell line MOLT-3, and early
myeloid leukemia cell line KG-1, both HDAC4 and HDAC9
CD were
co-expressed in these tissues (Fig. 5B, lanes 2,
14, and 17). To examine this further, we have
carried out RT-PCR analysis for multiple HDAC9 isoforms in a large
number of hematopoietic cell lines.
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Fig. 5.
Tissue distribution of HDAC9 transcripts.
A, the oligonucleotide primer sequences used for RT-PCR
analysis are detailed together with a schematic representation of the
primer positions in HDAC9 and HDAC9 CD (arrows).
B, differential expression of Class II HDACs in normal
tissues and leukemic cell lines. RT-PCR was performed on cDNA
derived from normal human tissue (1-13) and cell line (14-19) total
RNA as indicated. PCR products were resolved by agarose gel
electrophoresis and visualized by ethidium bromide under UV light.
C, HDAC9 isoforms are differentially expressed in the B cell
lineage and cell lines derived from B cell malignancies. RT-PCR was
performed on RNAs derived from normal human tissues (1-8), primary CLL
samples (9-12), primary childhood acute lymphoblastic leukemia samples
(13-16), or hematopoietic cell lines (17-41) total RNA, as indicated.
NLB (7-8) refers to normal B cell. Where indicated
(SAC/IL2), the cells were stimulated with
Staphylococcus aureus Cowan I strain (1/5000) and
interleukin 2 (50 units/ml). The identity of different isoforms
(indicated on the left) was confirmed by isoform-specific
and general oligonucleotide probes (data not shown).
CD transcripts are analyzed for expression of
exon 7 and 12 deletion splice variants in hematopoietic tissues and
cell lines (Fig. 5C), mRNA encoding the isoform lacking the catalytic domain appears to be expressed in many cell types. Although where co-expressed with full-length HDAC9, HDAC9
CD is considerably more abundant, we cannot exclude the possibility that this
may be due to the efficiency of amplification. In normal tissues, HDAC9
transcripts encoding the catalytic domain were found at low levels in
the bone marrow, spleen, and thymus (Fig. 5C, lanes
2-4). The highest levels, however, were observed in cells
expressing CD14+ve (monocyte/macrophage) and, to a lesser
extent, CD19+ve (B cell) surface markers (Fig.
5C, lanes 5 and 6). Further,
inspection of the RT-PCR data reveal that HDAC9 is generally expressed
in pre-B cell acute lymphoblastic leukemia cell lines (Fig.
5C, lanes 7-9), B cell lymphoma cell lines (Fig.
5C, lanes 10-13), and also the plasma cell line
U-266 (Fig. 5C, lane 14). HDAC9 is also expressed in some T cell lines (Fig. 5C, lanes 15-18).
HDAC9 is not expressed in various acute myeloid leukemia cell lines
(Fig. 5C, lanes 19-22), with the exception of
KG1 (a multilineage lymphomyelocytic cell line) (55), which expresses
HDAC9
CD. Two of four acute monocytic leukemia cell lines analyzed
expressed isoforms of HDAC9, and low levels are also found in the
erythroleukemia cell line, HEL (Fig. 5C, lane
27). Overall, the expression data suggest a selective although not
absolute bias of HDAC9 expression toward lymphoid and monocytic cells
within the hematopoietic system.
CD
transcripts containing exon 7.
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Fig. 6.
HDAC9 interacts in vivo and
in vitro with other histone deacetylases,
co-repressors, and transcription factors implicated in hematological
malignancies. A, GST fusion proteins were incubated with
35S-radiolabeled proteins as indicated, precipitated with
glutathione-Sepharose, and visualized by autoradiography. GST-HDAC9,
GST-HDAC9CD, and GST-HDAC9 CD were produced from Escherichia
coli DH5
. [35S]Methionine-labeled proteins were
synthesized in vitro using a rabbit reticulocyte
lysate-coupled transcription-translation system. B, the
indicated FLAG-tagged isoforms of HDAC9 were co-transfected with N-CoR
and TEL expression vectors. Total protein levels were normalized, and
protein complexes were immunoprecipitated with anti-N-CoR (C-20) and
anti-TEL (C-20) antibodies as indicated. Co-immunoprecipitated HDAC9
isoforms were resolved on a 5% SDS-polyacrylamide gel and detected by
immunoblotting with anti-FLAG M2 antibody. The arrowhead
indicates suspected degradation or incompletely translated products
that appear with the FLAG-tagged HDAC9 expression vectors containing
the catalytic domain. The cells transfected with pSG5 empty vector
(Stratagene) were included as a negative control (lanes 1 and 10). C, endogenous HDAC9 was
immunoprecipitated from NALM-6, RAJI, and REH cell lines with the
indicated antibodies. A specific blocking peptide for anti-N-CoR (C-20)
and an irrelevant antibody, anti-GAL4 (DBD) (RK5C1) were included as
negative controls. Samples containing co-immunoprecipitated HDAC9 were
resolved on a 7.5% SDS-polyacrylamide gel and detected by
immunoblotting with affinity-purified rabbit serum containing
antibodies raised against the C-terminal region of HDAC9.
exon7 compared with
N-CoR (Fig. 6B, lanes 13 and 18), indicating that the major site of interaction between TEL and HDAC9
lies within exon 7. Co-transfection of N-CoR or TEL with isoforms of
HDAC9 lacking the catalytic domain produced a markedly weaker
interaction relative to that observed for full-length HDAC9 when
compared with the in vitro data (Fig. 6B,
lanes 14, 15, 19, and 20).
This suggests that the catalytic domain may be interacting with other
proteins in vivo that act to stabilize the protein complex.
Finally, the interactions between endogenously expressed HDAC9 and
BCL-6, N-CoR, and TEL were examined in cell lines known to express
these proteins (40, 62, 63) (Fig. 6C). The results demonstrate that HDAC9 and the above factors can associate under physiological levels.
exon7 is completely
excluded from the nucleus. As previously reported for the mouse
homologue, HDAC9
CD punctate nuclear localization (64). The cellular
distribution of HDAC9 isoforms containing the catalytic domain was also
investigated (Fig. 7B) using an antibody raised against a
peptide corresponding to a C-terminal region of HDAC9 (see
"Experimental Procedures"). HDAC9 displayed a diffuse pattern of
distribution within the nuclei of the REH cells and was also found in
the cytoplasm, which corroborates RT-PCR data for this cell line
showing expression of HDAC9
exon7 mRNA (Fig. 5C,
lane 8).
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Fig. 7.
Immunofluorescence analysis of HDAC9.
A, HDAC9 is alternatively spliced to generate multiple
isoforms. COS-7 cells were transiently transfected with F-HDAC9,
F-HDAC9 exon7, F-HDAC9
exon12, F-HDAC9
exon15, F-HDAC9
CD, or
F-HDAC9
CD
exon7 as indicated. After methanol fixation, the cells
were stained with DAPI (blue) and FLAG antibody
(green). B, HDAC9 isoforms containing the
catalytic domain are expressed endogenously in the childhood acute
lymphoblastic leukemia cell line, REH. After fixation with 4%
paraformaldehyde, the cells were stained with To-pro-3 iodide
(blue) and affinity-purified anti-HDAC9 antibody.
C, HDAC9 interacts with the co-repressor N-CoR. COS-7 cells
were transiently transfected with N-CoR and F-HDAC9, F-HDAC9
exon7,
F-HDAC9
CD, or F-HDAC9
CD
exon7 as indicated. After methanol
fixation, the cells were stained with DAPI (blue) and
anti-FLAG M2 (green) or N-CoR (C-20) (red)
antibodies. D, HDAC9 interacts with TEL. The COS-7 cells
were transiently transfected TEL and F-HDAC9, F-HDAC9
exon7,
F-HDAC9
CD, or F-HDAC9
CD
exon7 as indicated. After methanol
fixation, the cells were stained with DAPI (blue) and
anti-FLAG M2 (green) or TEL (N-19) (red)
antibodies.
exon7, there was a
dramatic change in the distribution of the co-repressor to the
cytoplasmic location of the HDAC9
exon7 isoform. Recruitment or
retention of N-CoR to the cytoplasm was not observed upon co-expression with HDAC9
CD
exon7, indicating that the domains of interaction between HDAC9 and N-CoR lay within both exon 7 and the catalytic domain. This is supported by the in vitro data showing that
the catalytic domain of HDAC9 interacts with the C-terminal region of
N-CoR (Fig. 6A, lane 8). Similar results were
also observed for BCL-6 (data not shown). In contrast to N-CoR, TEL was
not excluded from the nucleus when co-expressed with either HDAC9
CD or the full-length HDAC9 lacking exon 7 (Fig. 7D), further
indicating that the main domain of interaction between HDAC9 and TEL is
mediated through exon 7. It appears from the above results that
different HDAC9 isoforms display individual interaction profiles with
various proteins, and this is reinforced by the observation that AML1 co-localizes exclusively with HDAC9
CD (data not shown), suggesting that the domain of interaction lies within the C-terminal sequence unique to this isoform.
CD, and HDAC9CD alone (Fig. 8A). A
GAL4uasx5-TK-Luc reporter gene was transiently transfected
into 293T cells together with the expression vectors for the indicated
GAL4 fusion proteins. Although HDAC9
CD lacks an HDAC domain, it
associates with other HDACs and co-repressors (Figs. 6 and 7), and this
may be reflected in its ability to repress reporter gene
expression.
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Fig. 8.
HDAC9 represses transcriptional activity
in vivo. A, GAL4-HDAC9 represses basal transcription.
GAL4uasx5-TK-Luc (400 ng) was transfected into 293T cells
along with GAL4DBD-HDAC9, -HDAC9 CD, or -HDAC9CD (200 ng each) as
indicated. Luciferase activity was normalized by co-transfection of
-galactosidase. The parent GAL4 fusion vector, pM
(Clontech), was used to equalize the amount of
GAL4DBD in each transfection. The basal value was set to 1. B, HDAC9 represses MEF2D-mediated activation.
MEF2REx3-Tk-Luc (200 ng) was transfected into 293T cells
along with pSG5 empty vector, pCMV-MEF2D, F-HDAC9, or F-HDAC9
CD/MITR
(200 ng each). pSG5 empty vector (Stratagene) was used to equalize the
amount of DNA in each transfection. The value for
MEF2REx3-Tk-Luc alone was set to 1. C, HDAC9
enhances TEL-mediated repression. TELREx3-Tk-Luc (400 ng)
was transfected into 293T cells along with pSG5 empty vector, pSG5-TEL,
F-HDAC9, or F-HDAC9
CD/MITR (200 ng each). The value for
TELREx3-Tk-Luc alone was set to 1. The values are shown as
fold repression.
CD (Fig.
8B).
CD, they reported that it
associated with myocyte enhancer factor 2D (xMEF2D) and repressed
xMEF2D-mediated transcription. In the human hematopoietic system, MEF2D
is found in both B and T cells (65), and HDAC9 was tested for its
ability to affect MEF2D-mediated transcription. As expected,
co-transfection of HDAC9 with MEF2D abolished MEF2D-mediated
transcriptional activation and repressed a
MEF2REx3-Tk-Luc reporter. Co-transfection of HDAC9
CD
with MEF2D produces a similar result, but the level of MEF2D-mediated
activation was only returned to the basal level (Fig. 8C).
This reflects the results observed with TEL promoter activity and
indicates that full-length HDAC9 forms a more powerful repressor
complex in this context.
exon12 isoform, which only encodes 80 residues, shows an
unexpectedly large decrease in apparent size (relative to
7 and
15 deletions), suggesting the presence of a site of
post-translational modification. This was thought most likely to be
sumoylation, which has been detected on the corresponding region of
HDAC4 (46, 53). To examine this possibility, tsCOS cells were
transfected with HDAC9 and HDAC9
exon12 together with polyhistidine-tagged SUMO-1 and -2. Upon nickel affinity precipitation of His-tagged protein complexes, only the full-length HDAC9 isoform containing exon 12 was visible as a SUMO-1 or -2 conjugate (Fig. 10, lanes 8 and
9). SUMO-2 may itself be sumoylated to form polymeric chains
(53), and the multiple bands observed in lane 8 are due to
endogenous SUMO-2 present in the cells. This is illustrated by the fact
that the polymeric chains containing His-tagged SUMO-2 in lane
9 migrate slightly more slowly than those observed in lane
8, where only the terminal SUMO-1 may be His-tagged. Note that
mono-sumoylated HDAC9 precipitates with markedly less efficiency compared with di- and tri-sumoylated HDAC9. This is most likely due to
a combination the presence of multiple His tags and/or improved access
to the tag.
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Fig. 9.
Expression of endogenous HDAC9 proteins.
Whole cell lysates were prepared from tsCOS cells transfected with
F-HDAC9, F-HDAC9 exon7, F-HDAC9
exon12, and selected cell lines as
indicated. Total protein levels were normalized, and the samples were
resolved on a 5% SDS-polyacrylamide gel. Western blot analysis was
performed with affinity-purified rabbit serum containing antibodies
raised against the C-terminal region of HDAC9. Enhanced
chemiluminescence was used for detection.
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Fig. 10.
SUMO-1 and -2 are conjugated to HDAC9 but
not HDAC9 exon12 in vivo.
Whole cell lysates were prepared from tsCOS cells co-transfected with
F-HDAC9, F- HDAC9
exon12, and His6-tagged SUMO-1 or -2 as
indicated. pSG5 empty vector (Stratagene) was used as a control
plasmid. Samples precipitated with trichloroacetic acid to show input
protein levels (lanes 1-6) and nickel pull-downs
(lanes 7-12) were analyzed by Western blotting using
anti-HDAC9 rabbit polyclonal antibody. The open arrowheads
indicate mono-, di-, and tri-sumoylated HDAC9. The solid
arrowhead refers to a nonspecific band observed with the
trichloroacetic acid precipitates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD. Recent studies on HDAC4 have shown that the region encoded
by exon 7 contains residues that form a powerful nuclear localization
signal and two serines, which when phosphorylated facilitate the
binding of 14-3-3 signaling proteins (52). These residues are conserved
in HDAC9, and proteins lacking the exon7 region may have a dramatic
effect on the cellular localization of interacting factors such as
N-CoR (Fig. 7C). The interactions between HDAC9 isoforms and
interacting proteins such as BCl-6, TEL, or N-CoR merit more detailed
investigation, but it seems likely that many of the interactions are
mediated through multiple regions in HDAC9 that are specific for a
given partner protein. This introduces a level of complexity with
regard to HDAC9 association with partner proteins not thus far reported for other family members. It has been suggested that HDACs may act as
recruiting centers for specific protein complexes as well as being
functional enzymes (79), and these data would tend to be in line with
this notion.
exon12, which also contains potential functional domains such as a leucine zipper motif and a sumoylation site. The HDAC9
exon12 isoform cannot be sumoylated and may prove to exert an important and
separate function because SUMO is believed to alter the interaction properties of its targets, often affecting their localization within
the cell (80). Moreover, recent studies have shown that sumoylation can
affect HDAC1 and HDAC4 catalytic activity (46, 81).
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ACKNOWLEDGEMENTS |
---|
We thank Eric Verdin for HDAC3 cDNA, Stuart Schreiber and Tony Kouzarides for HDAC4 cDNAs, Eric Oslon for MEF2D cDNA, Riccardo Dalla-Favera for BCL-6 cDNA, and Anne Dejean for SUMO-1 and -2 cDNA.
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FOOTNOTES |
---|
* This work was supported by funds from the Leukemia Research Fund of Great Britain, the Institute of Cancer Research Studentship (to K. P.), the Kay Kendall Leukemia Fund (to F. G.), and the Samuel Waxman Cancer Research Foundation.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/EBI Data Bank with accession number(s) AY197371.
¶ To whom correspondence should be addressed: Inst. of Cancer Research, 237 Fulham Rd., London SW3 6JB, UK. Tel.: 44-207-3528133; Fax: 44-207-3523299; E-mail: a.zelent@icr.ac.uk.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M212935200
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ABBREVIATIONS |
---|
The abbreviations used are: HDAC, histone deacetylase; MITR, MEF2-interacting transcriptional repressor; HDRP, histone deacetylase-related protein; GST, glutathione S-transferase; Luc, luciferase; PBS, phosphate-buffered saline; RT, reverse transcriptase; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4',6'-diamidino-2-phenylindole hydrochloride; contig, group of overlapping clones; ORF, open reading frame; DBD, DNA-binding domain.
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REFERENCES |
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---|
1. | Wu, J., and Grunstein, M. (2000) Trends Biochem. Sci. 25, 619-623[CrossRef][Medline] [Order article via Infotrieve] |
2. | Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E., and Moudrianakis, E. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10148-10152[Abstract] |
3. | Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Nature 389, 251-260[CrossRef][Medline] [Order article via Infotrieve] |
4. | Grunstein, M. (1997) Nature 389, 349-352[CrossRef][Medline] [Order article via Infotrieve] |
5. | Cress, W. D., and Seto, E. (2000) J. Cell. Physiol. 184, 1-16[CrossRef][Medline] [Order article via Infotrieve] |
6. | Gray, S. G., and Ekstrom, T. J. (2001) Exp. Cell Res. 262, 75-83[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Zhou, X.,
Marks, P. A.,
Rifkind, R. A.,
and Richon, V. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
10572-10577 |
8. |
Kao, H. Y.,
Lee, C. H.,
Komarov, A.,
Han, C. C.,
and Evans, R. M.
(2002)
J. Biol. Chem.
277,
187-193 |
9. |
Gao, L.,
Cueto, M. A.,
Asselbergs, F.,
and Atadja, P.
(2002)
J. Biol. Chem.
277,
25748-25755 |
10. |
Emiliani, S.,
Fischle, W.,
Van Lint, C.,
Al-Abed, Y.,
and Verdin, E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2795-2800 |
11. |
Yang, W. M.,
Yao, Y. L.,
Sun, J. M.,
Davie, J. R.,
and Seto, E.
(1997)
J. Biol. Chem.
272,
28001-28007 |
12. |
Hu, E.,
Chen, Z.,
Fredrickson, T.,
Zhu, Y.,
Kirkpatrick, R.,
Zhang, G. F.,
Johanson, K.,
Sung, C. M.,
Liu, R.,
and Winkler, J.
(2000)
J. Biol. Chem.
275,
15254-15264 |
13. |
Grozinger, C. M.,
Hassig, C. A.,
and Schreiber, S. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4868-4873 |
14. |
Verdel, A.,
and Khochbin, S.
(1999)
J. Biol. Chem.
274,
2440-2445 |
15. |
Kao, H. Y.,
Downes, M.,
Ordentlich, P.,
and Evans, R. M.
(2000)
Genes Dev.
14,
55-66 |
16. |
Fischle, W.,
Emiliani, S.,
Hendzel, M. J.,
Nagase, T.,
Nomura, N.,
Voelter, W.,
and Verdin, E.
(1999)
J. Biol. Chem.
274,
11713-11720 |
17. |
Fischle, W.,
Dequiedt, F.,
Fillion, M.,
Hendzel, M. J.,
Voelter, W.,
and Verdin, E.
(2001)
J. Biol. Chem.
276,
35826-35835 |
18. |
Fischer, D. D.,
Cai, R.,
Bhatia, U.,
Asselbergs, F. A.,
Song, C.,
Terry, R.,
Trogani, N.,
Widmer, R.,
Atadja, P.,
and Cohen, D.
(2002)
J. Biol. Chem.
277,
6656-6666 |
19. |
Guardiola, A. R.,
and Yao, T. P.
(2002)
J. Biol. Chem.
277,
3350-3356 |
20. |
Tong, J. J.,
Liu, J.,
Bertos, N. R.,
and Yang, X. J.
(2002)
Nucleic Acids Res.
30,
1114-1123 |
21. |
Grozinger, C. M.,
and Schreiber, S. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7835-7840 |
22. |
McKinsey, T. A.,
Zhang, C. L.,
and Olson, E. N.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
14400-14405 |
23. | McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000) Nature 408, 106-111[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Wang, A. H.,
Kruhlak, M. J.,
Wu, J.,
Bertos, N. R.,
Vezmar, M.,
Posner, B. I.,
Bazett-Jones, D. P.,
and Yang, X. J.
(2000)
Mol. Cell. Biol.
20,
6904-6912 |
25. |
Zhao, X.,
Ito, A.,
Kane, C. D.,
Liao, T. S.,
Bolger, T. A.,
Lemrow, S. M.,
Means, A. R.,
and Yao, T. P.
(2001)
J. Biol. Chem.
276,
35042-35048 |
26. | Alland, L., Muhle, R., Hou, H. J., Potes, J., Chin, L., Schreiber-Agus, N., and DePinho, R. A. (1997) Nature 387, 49-55[CrossRef][Medline] [Order article via Infotrieve] |
27. | Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 43-48[CrossRef][Medline] [Order article via Infotrieve] |
28. | Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997) Cell 89, 373-801[Medline] [Order article via Infotrieve] |
29. | Hassig, C. A., Fleischer, T. C., Billin, A. N., Schreiber, S. L., and Ayer, D. E. (1997) Cell 89, 341-371[Medline] [Order article via Infotrieve] |
30. | Laherty, C. D., Yang, W. M., Sun, J. M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997) Cell 89, 349-356[Medline] [Order article via Infotrieve] |
31. |
Huang, E. Y.,
Zhang, J.,
Miska, E. A.,
Guenther, M. G.,
Kouzarides, T.,
and Lazar, M. A.
(2000)
Genes Dev.
14,
45-54 |
32. | Ng, H. H., and Bird, A. (2000) Trends Biochem. Sci. 25, 121-126[CrossRef][Medline] [Order article via Infotrieve] |
33. | Fischle, W., Kiermer, V., Dequiedt, F., and Verdin, E. (2001) Biochem. Cell Biol. 79, 337-348[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Guenther, M. G.,
Barak, O.,
and Lazar, M. A.
(2001)
Mol. Cell. Biol.
21,
6091-6101 |
35. |
Sparrow, D. B.,
Miska, E. A.,
Langley, E.,
Reynaud-Deonauth, S.,
Kotecha, S.,
Towers, N.,
Spohr, G.,
Kouzarides, T.,
and Mohun, T. J.
(1999)
EMBO J.
18,
5085-5098 |
36. | Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1998) DNA Res. 5, 277-286[Medline] [Order article via Infotrieve] |
37. |
Zhou, X.,
Richon, V. M.,
Rifkind, R. A.,
and Marks, P. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1056-1061 |
38. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology , Green Publishing Associates and Wiley-Interscience, New York |
39. | Nordeen, S. K. (1988) BioTechniques 6, 454-458[Medline] [Order article via Infotrieve] |
40. |
Guidez, F.,
Petrie, K.,
Ford, A. M.,
Lu, H.,
Bennett, C. A.,
MacGregor, A.,
Hannemann, J.,
Ito, Y.,
Ghysdael, J.,
Greaves, M.,
Wiedemann, L. M.,
and Zelent, A.
(2000)
Blood
96,
2557-2561 |
41. | Ye, B. H., Lista, F., Lo Coco, F., Knowles, D. M., Offit, K., Chaganti, R. S., and Dalla-Favera, R. (1993) Science 262, 747-750[Medline] [Order article via Infotrieve] |
42. | Martin, J. F., Miano, J. M., Hustad, C. M., Copeland, N. G., Jenkins, N. A., and Olson, E. N. (1994) Mol. Cell. Biol. 14, 1647-1656[Abstract] |
43. |
Dong, S.,
Zhu, J.,
Reid, A.,
Strutt, P.,
Guidez, F.,
Zhong, H.-J.,
Wang, Z.-Y.,
Licht, J.,
Waxman, S.,
Chomienne, C.,
Chen, Z.,
Zelent, A.,
and Chen, S.-J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3624-3629 |
44. |
Soderstrom, M.,
Vo, A.,
Heinzel, T.,
Lavinsky, R. M.,
Yang, W. M.,
Seto, E.,
Peterson, D. A.,
Rosenfeld, M. G.,
and Glass, C. K.
(1997)
Mol. Endocrinol.
11,
682-692 |
45. | Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) Science 272, 408-411[Abstract] |
46. |
Kirsh, O.,
Seeler, J. S.,
Pichler, A.,
Gast, A.,
Muller, S.,
Miska, E.,
Mathieu, M.,
Harel-Bellan, A.,
Kouzarides, T.,
Melchior, F.,
and Dejean, A.
(2002)
EMBO J.
21,
2682-2691 |
47. | Rio, D. C., Clark, S. G., and Tjian, R. (1985) Science 227, 23-28[Medline] [Order article via Infotrieve] |
48. | Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D., and Broach, J. R. (1993) Genes Dev. 7, 592-604[Abstract] |
49. |
Seeler, J. S.,
Marchio, A.,
Losson, R.,
Desterro, J. M. P.,
Hay, R. T.,
Chambon, P.,
and Dejean, A.
(2001)
Mol. Cell. Biol.
21,
3314-3324 |
50. |
Benson, D. A.,
Karsch-Mizrachi, I.,
Lipman, D. J.,
Ostell, J.,
Rapp, B. A.,
and Wheeler, D. L.
(2002)
Nucleic Acids Res.
30,
17-20 |
51. |
Hubbard, T.,
Barker, D.,
Birney, E.,
Cameron, G.,
Chen, Y.,
Clark, L.,
Cox, T.,
Cuff, J.,
Curwen, V.,
Down, T.,
Durbin, R.,
Eyras, E.,
Gilbert, J.,
Hammond, M.,
Huminiecki, L.,
Kasprzyk, A.,
Lehvaslaiho, H.,
Lijnzaad, P.,
Melsopp, C.,
Mongin, E.,
Pettett, R.,
Pocock, M.,
Potter, S.,
Rust, A.,
Schmidt, E.,
Searle, S.,
Slater, G.,
Smith, J.,
Spooner, W.,
Stabenau, A.,
Stalker, J.,
Stupka, E.,
Ureta-Vidal, A.,
Vastrik, I.,
and Clamp, M.
(2002)
Nucleic Acids Res.
30,
38-41 |
52. |
Wang, A. H.,
and Yang, X. J.
(2001)
Mol. Cell. Biol.
21,
5992-6005 |
53. |
Tatham, M. H.,
Jaffray, E.,
Vaughan, O. A.,
Desterro, J. M.,
Botting, C. H.,
Naismith, J. H.,
and Hay, R. T.
(2001)
J. Biol. Chem.
276,
35368-35374 |
54. |
Zhang, C. L.,
McKinsey, T. A.,
Lu, J. R.,
and Olson, E. N.
(2001)
J. Biol. Chem.
276,
35-39 |
55. | Furley, A. J., Reeves, B. R., Mizutani, S., Altass, L. J., Watt, S. M., Jacob, M. C., van den Elsen, P., Terhorst, C., and Greaves, M. F. (1986) Blood 68, 1101-1107[Abstract] |
56. | He, L. Z., Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., Zelent, A., and Pandolfi, P. P. (1998) Nat. Genet. 18, 126-135[Medline] [Order article via Infotrieve] |
57. |
Gelmetti, V.,
Zhang, J.,
Fanelli, M.,
Minucci, S.,
Pelicci, P. G.,
and Lazar, M. A.
(1998)
Mol. Cell. Biol.
18,
7185-7191 |
58. |
Romana, S. P.,
Mauchauffe, M.,
Le Coniat, M.,
Chumakov, I.,
Le Paslier, D.,
Berger, R.,
and Bernard, O. A.
(1995)
Blood
85,
3662-3670 |
59. | Golub, T. R., Barker, G. F., Bohlander, S. K., Hiebert, S. W., Ward, D. C., Bray-Ward, P., Morgan, E., Raimondi, S. C., Rowley, J. D., and Gilliland, D. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4917-4921[Abstract] |
60. |
Capello, D.,
Vitolo, U.,
Pasqualucci, L.,
Quattrone, S.,
Migliaretti, G.,
Fassone, L.,
Ariatti, C.,
Vivenza, D.,
Gloghini, A.,
Pastore, C.,
Lanza, C.,
Nomdedeu, J.,
Botto, B.,
Freilone, R.,
Buonaiuto, D.,
Zagonel, V.,
Gallo, E.,
Palestro, G.,
Saglio, G.,
Dalla-Favera, R.,
Carbone, A.,
and Gaidano, G.
(2000)
Blood
95,
651-659 |
61. | Guidez, F., and Zelent, A. (2001) Curr. Top. Microbiol. Immunol. 254, 165-185[Medline] [Order article via Infotrieve] |
62. |
Allman, D.,
Jain, A.,
Dent, A.,
Maile, R. R.,
Selvaggi, T.,
Kehry, M. R.,
and Staudt, L. M.
(1996)
Blood
87,
5257-5268 |
63. | Agape, P., Gerard, B., Cave, H., Devaux, I., Vilmer, E., Lecomte, M. C., and Grandchamp, B. (1997) Br. J. Haematol. 98, 234-239[CrossRef][Medline] [Order article via Infotrieve] |
64. |
Zhang, C. L.,
McKinsey, T. A.,
and Olson, E. N.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
7354-7359 |
65. | Powlesland, R. M., Charles, A. K., Malik, K. T., Reynolds, P. A., Pires, S., Boavida, M., and Brown, K. W. (2000) Br. J. Cancer 82, 323-329[CrossRef][Medline] [Order article via Infotrieve] |
66. | Tsuji, K., Narahara, K., Kikkawa, K., Murakami, M., Yokoyama, Y., Ninomiya, S., and Seino, Y. (1994) Am. J. Med. Genet. 49, 98-102[Medline] [Order article via Infotrieve] |
67. | Lewanda, A. F., Green, E. D., Weissenbach, J., Jerald, H., Taylor, E., Summar, M. L., Phillips, J. A., 3rd, Cohen, M., Feingold, M., Mouradian, W., Clarren, S. K., and Wang Jabs, E. (1994) Am. J. Hum. Genet. 55, 1195-1201[Medline] [Order article via Infotrieve] |
68. | Johnson, D., Horsley, S. W., Moloney, D. M., Oldridge, M., Twigg, S. R., Walsh, S., Barrow, M., Njolstad, P. R., Kunz, J., Ashworth, G. J., Wall, S. A., Kearney, L., and Wilkie, A. O. (1998) Am. J. Hum. Genet. 63, 1282-1293[CrossRef][Medline] [Order article via Infotrieve] |
69. | Stankiewicz, P., Thiele, H., Baldermann, C., Kruger, A., Giannakudis, I., Dorr, S., Werner, N., Kunz, J., Rappold, G. A., and Hansmann, I. (2001) Am. J. Med. Genet. 103, 56-62[CrossRef][Medline] [Order article via Infotrieve] |
70. | Aragane, H., Sakakura, C., Nakanishi, M., Yasuoka, R., Fujita, Y., Taniguchi, H., Hagiwara, A., Yamaguchi, T., Abe, T., Inazawa, J., and Yamagishi, H. (2001) Int. J. Cancer 94, 623-629[CrossRef][Medline] [Order article via Infotrieve] |
71. | Schmidt, H., Taubert, H., Wurl, P., Kappler, M., Lange, H., Bartel, F., Bache, M., Holzhausen, H. J., and Hinze, R. (2002) Genes Chromosomes Cancer 34, 69-77[CrossRef][Medline] [Order article via Infotrieve] |
72. | Jarosova, M., Holzerova, M., Jedlickova, K., Mihal, V., Zuna, J., Stary, J., Pospisilova, D., Zemanova, Z., Trka, J., Blazek, J., Pikalova, Z., and Indrak, K. (2000) Cancer Genet. Cytogenet. 123, 114-122[CrossRef][Medline] [Order article via Infotrieve] |
73. | Schmidt, H., Wurl, P., Taubert, H., Meye, A., Bache, M., Holzhausen, H. J., and Hinze, R. (1999) Genes Chromosomes Cancer 25, 205-211[CrossRef][Medline] [Order article via Infotrieve] |
74. |
Krebs, I.,
Weis, I.,
Hudler, M.,
Rommens, J. M.,
Roth, H.,
Scherer, S. W.,
Tsui, L. C.,
Fuchtbauer, E. M.,
Grzeschik, K. H.,
Tsuji, K.,
and Kunz, J.
(1997)
Hum. Mol. Genet.
6,
1079-1086 |
75. | el Ghouzzi, V., Le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A. L., Munnich, A., and Bonaventure, J. (1997) Nat. Genet. 15, 42-46[Medline] [Order article via Infotrieve] |
76. | Howard, T. D., Paznekas, W. A., Green, E. D., Chiang, L. C., Ma, N., Ortiz de Luna, R. I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A. D., and Jabs, E. W. (1997) Nat. Genet. 15, 36-41[Medline] [Order article via Infotrieve] |
77. | Mahlknecht, U., Schnittger, S., Ottmann, O. G., Schoch, C., Mosebach, M., Hiddemann, W., and Hoelzer, D. (2000) Biochim. Biophys. Acta 1493, 342-348[Medline] [Order article via Infotrieve] |
78. | Mahlknecht, U., Emiliani, S., Najfeld, V., Young, S., and Verdin, E. (1999) Genomics 56, 197-202[CrossRef][Medline] [Order article via Infotrieve] |
79. | Khochbin, S., and Kao, H. Y. (2001) FEBS Lett. 494, 141-144[CrossRef][Medline] [Order article via Infotrieve] |
80. | Seeler, J. S., and Dejean, A. (2001) Oncogene 20, 7243-7249[CrossRef][Medline] [Order article via Infotrieve] |
81. | David, G., Neptune, M. A., and DePinho, R. A. (2002) J. Biol. Chem. 17, 17 |
82. | Black, D. L. (2000) Cell 103, 367-370[Medline] [Order article via Infotrieve] |
83. | Graveley, B. R. (2001) Trends Genet 17, 100-107[CrossRef][Medline] [Order article via Infotrieve] |
84. |
Kent, W. J.
(2002)
Genome Res.
12,
656-664 |
85. | Enver, T., Brewer, A. C., and Patient, R. K. (1985) Nature 318, 680-683[Medline] [Order article via Infotrieve] |