From the Laboratoire de Biologie Moléculaire du Cycle Cellulaire, INSERM U309, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 La Tronche Cedex, France
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ABSTRACT |
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The histone deacetylase domain of almost all
members of higher eukaryotic histone deacetylases already identified
(HDAC family) is highly homologous to that of yeast RPD3. In this paper
we report the cloning of two cDNAs encoding members of a new family
of histone deacetylase in mouse that show a better homology to yeast
HDA1 histone deacetylase. These cDNAs encode relatively large
proteins, presenting an in vitro trichostatin A-sensitive
histone deacetylase activity. Interestingly, one, mHDA2, encodes a
protein with two putative deacetylase domains, and the other, mHDA1,
contains only one deacetylase homology domain, located at the
C-terminal half of the protein. Our data showed that these newly
identified genes could belong to a network of genes coordinately
regulated and involved in the remodeling of chromatin during cell
differentiation. Indeed, the expression of mHDA1 and mHDA2 is tightly
linked to the state of cell differentiation, behaving therefore like
the histone H1°-encoding gene. Moreover, like histone H1° gene,
mHDA1 and mHDA2 gene expression is induced upon deacetylase inhibitor treatment. We postulate the existence of a regulatory mechanism, commanding a coordinate expression of a group of genes involved in the
remodeling of chromatin not only during cell differentiation but also
after abnormal histone acetylation.
Enzymes involved in the modification of histones by acetylation,
histone acetyltransferases
(HAT)1 and histone
deacetylases (HD), are believed to play an important role in the
regulation of transcription (1, 2). Moreover, acetylation appears to be
a signaling process that involves not only histones but also
non-histone proteins, mostly transcriptional regulators (3, 4). The
identification of HATs and HDs are therefore crucial for the full
understanding of the function of the signaling pathways through
acetylation. The first histone deacetylase identified, HDAC1, showed a
striking sequence homology with a yeast protein, RPD3 (5). After this
discovery, not only histone deacetylases have been identified in many
higher eukaryotes, but also variants have been reported (6-8).
Sequence analysis performed on these proteins showed that they all
contain a conserved feature; a large domain of homology with the yeast
RPD3, covering the 2/3 N-terminal of the protein and a short C-terminal
region with the most variable sequence (9). The RPD3 homology domain is
also shared by several procaryotic proteins interacting with various
acetylated substrates (9-11). These observations strongly suggested
that this homology domain is also the deacetylase domain (9). Recently,
mutagenesis of this domain confirmed its involvement in the enzymatic
activity (12). Biochemical study in yeast allowed the identification
(13) and the cloning of another histone deacetylase, HDA1 (14). Indeed,
in yeast, two histone deacetylase complexes have been functionally
identified (although, based on sequence homology, other potential
members have been identified (14)): one, containing the histone
deacetylase RPD3 (HDB complex), and the other (HDA complex), containing
a distinct member of the histone deacetylase family, the HDA1. The
protein is functionally different from RPD3 as it shows a greater
sensitivity to trichostatin A (TSA) and is present in a complex
different from that containing RPD3 (14). Moreover, HDA1 is not
involved in the transcriptional regulation of genes controlled by RPD3
(15). Interestingly, the deacetylase homology domain of this protein is
homologous but significantly different from that of RPD3 (14). The
putative histone deacetylase domain of all the histone deacetylases
reported in higher eukaryotes, except that of maize nucleolar HD2 (16), show striking sequence homology to yeast RPD3 (9-11), suggesting that
they are all members of the same family. It was therefore very tempting
to know if, as in yeast, higher eukaryote deacetylases with divergent
sequence in their deacetylase domain can be demonstrated. To identify
members of such a family in higher eukaryotes, we sought a strategy
based on the systematic search of expressed sequence tag (EST) data
base for open reading frames, showing a better homology to yeast HDA1
than to yeast RPD3. This screening yields a considerable mass of raw
information that has been treated, thanks to a computer program we
designed to organize the information in a way that allows a rapid
identification of interesting ESTs (consultable on an Internet site,
see below). This strategy allowed us to clone two members of this
family in mouse. The histone deacetylase activity of these proteins was
confirmed, and the study of their expression allowed us to demonstrate
the existence of a network of genes involved in the organization of
chromatin and the control of cell differentiation that shows a
coordinate profile of expression.
Identification of Higher Eukaryotic Homologues of Yeast HDA1
Data base EST and data base EST cumulative updates were searched
using the BCM search launcher Nucleic Acid Sequence Searches facility
at NCBI2 through the
TBLASTX/dbest that allows a search for homology considering the six
translation frames of the query versus the six translation frames of ESTs. Although the reading frame of the query is known, we
searched with the six possible frames as control to know if incorrect
frames give rise to significant hits. In almost all the cases, the
program found significant hits only with the correct frame of the query.
The result of this search, carried out with the yeast HDA1 sequence and
26 other chromatin/transcription-related yeast proteins, can be
consulted.3 To rapidly
identify interesting ESTs (those presenting the best homology to the
most N-terminal part of yeast HDA1), we designed a computer program to
treat the raw data and to set up a schematic representation of
homologous ESTs considering all the homology parameters (see below).
Homologous EST sequences were presented as a bar under the schematic
representation of the query (yeast HDA1). The bar indicates the largest
homologous fragment possible, built from the actual sequence alignments
output. Moreover, for each EST, we considered the homology score and
the number of homologous sub-fragments (n) and
p(n) (see BLAST server). When n is greater than
1, union of the fragment showing the highest score (the highest scoring
segment pair (HSP)) and other segment pairs (lower scoring pair or
(LSP)), was performed. In case of discontinued homologies, the gap
between two fragments was symbolized in gray. To have the most
significant representation possible, we listed ESTs having a high score
greater than 100. This methodology allowed us to identify two different
mouse ESTs presenting an open reading frames with significant sequence
homology with yeast HDA1. These ESTs were purchased from Genom System
Inc. and sequenced.
Cloning of Full-length mHDA1 and mHDA2 cDNAs
To obtain the complete mHDA1 and mHDA2 coding sequence, we used
a 5' rapid amplification of cDNA ends-based strategy. A
Marathon-Ready mouse cDNA library (CLONTECH)
was purchased, and amplifications were performed using two nested
cDNA-specific primers and a primer corresponding to the linker
sequence following the user manual protocol. The polymerase chain
reaction products were then cloned and sequenced.
Immunoprecipitation Histone Deacetylase Assays
cDNAs containing the whole mHDA1 and mHDA2 coding region
were cloned in the pcDNA-His vector (Invitrogen). As a control, we also cloned the cDNA encoding HDAC1 (an EST purchased from Genome system Inc. and entirely sequenced) in the same vector. Rat embryonic cells (17) were transfected with 10 µg of each plasmid using Lipofectin reagent (Life Technologies, Inc.). 24 h after
transfection, cells were harvested and sonicated in the lysis buffer
(100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1%
Nonidet P-40, and protease inhibitor mixture: Boehringer Mannheim).
Protein G-Sepharose was added to the cleared lysate for 1 h at
4 °C. After the removal of the beads, 2 µg of anti-Xpress antibody
(Invitrogen) and 2 µg of anti-penta-His antibody (Qiagen) were added
to the lysate, and the incubation was continued overnight.
Immunocomplexes were precipitated using protein G-Sepharose and washed
three times in TL1 (100 mM Tris HCl, pH 8, 0.5 M LiCl, 0.5% Nonidet P-40) and three times in HD buffer
(75 mM Tris-HCl, pH 7, 275 mM NaCl, 0.1 mM EDTA). Finally, the beads containing the immunocomplexes
were resuspended in 100 µl of HD buffer, and 15 µl portions were
assayed for enzymatic activity. 3H-labeled acetylated
histones were obtained according to the protocol published by Carmen
et al. (13). Each assay was performed in duplicate in 50 µl containing 60,000 cpm [3H]acetate-labeled histones
and immunocomplexes in the presence or absence of 100 ng/ml TSA.
Incubation was carried out for 2 h at room temperature, the volume
was adjusted to 200 µl with HD buffer, and 50 µl of Stop buffer
(13) were added. The released [3H]acetic acid was
extracted with 600 µl of ethyl acetate and measured. Cpm
corresponding to the nonenzymatic release of [3H]acetate
(obtained by incubating 3H-labeled histones with protein
G-Sepharose in HD buffer, around 300 cpm) were subtracted from values
obtained with the immunocomplexes.
Cell Culture Conditions
Murine erythroleukemia cells and murine B16 cells were
maintained in culture and induced to differentiate as described
previously (18, 19). FM3A and TR303 cells were cultured and treated
with trichostatin A as described previously (20).
Analysis of Histone Hyperacetylation after Trichostatin A
Treatment
FM3A and TR303 cell lines were treated with different
concentrations of trichostatin A for 6 h. Cells were lysed, and
histones were purified and analyzed on a Triton/acid/urea gel as
described previously (20). The appearance of hyperacetylated H4 was
also monitored using antibody raised against hyperacetylated histone H4
(Upstate Biotechnology Inc.) followed by cytofluorimetric measurement of immunofluorescence according to the published protocol (21).
Northern Blot Analysis
Cells in Culture--
Total RNA was purified from MEL-, B16, and
FM3A/TR303 cells after various durations of treatment by inducer using
Tri-reagent (Sigma).
Rat Partial Hepatectomy and RNA Preparation--
Male Wistar
rats were hepatectomized, and RNA was purified from control liver or
from liver different times after the surgery exactly as described by
Khochbin et al. (22).
Adult Mouse Tissues--
A mouse multiple tissue Northern blot
was obtained from CLONTECH and analyzed using
different probes as mentioned in the text.
Cloning of Mouse Homologues of Yeast HDA1--
ESTs encoding
potential homologues of yeast HDA1 (yHDA1) were identified as described
under "Materials and Methods." Although ESTs encoding HDA1
homologous proteins were found from human, mouse,
Drosophila, and Caenorhabditis elegans (see the
web site), we decided to continue our work on mouse. Two ESTs
potentially encoding two distinct yHDA1 homologues were obtained and
sequenced. We named these clones mHDA1 and mHDA2, and the length of the
sequence obtained was 1000 base pairs for mHDA1 and 1782 for mHDA2.
Both contained the 3'-untranslated region and an open reading frame showing a convincing homology to yHDA1. These cDNAs were used as
probes, and both revealed a band approximately 4 kilobases long in a
Northern blot containing RNA from different mouse tissues. Therefore,
around 2 to 3 additional kilobase pairs of cDNA had to be cloned
and sequenced. A 5' rapid amplification of cDNA ends cloning
strategy allowed us to determine the sequence of the missing 5' part of
the two cDNAs. The sequence analysis showed that mHDA1 and mHDA2
encode proteins of 991 and 1025 amino acids, respectively. A data base
search showed that the best homology found is with yHDA1. The yHDA
homology domain of mHDA1 is located in the C-terminal half of the
protein (Fig. 1A) and shows
about 42% amino acid identity with yHDA1 over 290 residues (Fig.
1B). The remaining N-terminal part of the protein, separated
by a polyglutamic stretch from the deacetylase homology domain, does
not show any significant homology with known proteins. The yHDA1
homology domain of mHDA2 covers two distinct portions of the protein
(Fig. 1A). The first region is located in the N-terminal
half and shows 40% amino acids identity over 273 residues. The second
region, separated from the first one by a glutamate-rich stretch, is
located in the C-terminal half of the protein and is 45% identical to
yHDA1 over 314 amino acids (Fig. 1B).
These HDA1 homology domains are themselves homologous to yRPD3 (36%
identity for mHDA1 and 30% and 27% for the domain 1 and 2 of mHDA2,
respectively). Importantly, residues shown to be essential for
deacetylase activity of HDAC1/RPD3 (histidines and aspartic acids, see
Ref. 12) are strictly conserved (Fig. 1B). These observations strongly suggest that the mHDA1 and mHDA2 homology domains
are actually deacetylase domains and that mHDA2 has two distinct
deacetylase domains.
mHDA1 and mHDA2 Are Histone Deacetylases--
The deacetylase
homology domains of mHDA1 and mHDA2 present all the sequence features
shared by enzymes interacting with acetylated substrates (all the amino
acids present in procaryotic acetion utilization protein, acuC,
acetylpolyamine amidohydrolase, and HDAC members (9-11) are also
present in these proteins). The fact that in eukaryotes, these enzymes
are histone deacetylases strongly suggests that mHDA1 and mHDA2 are
also histone deacetylases. We therefore set up experiments to show the
histone deacetylase activity of these new members. The entire coding
region of mHDA1 and mHDA2 as well as that of HDAC1 were cloned in
pcDNA-His expression vector. mHDA1 and mHDA2 are able to be
expressed efficiently in vitro and in vivo (Fig.
2A). After transfection of
cells with these constructs, tagged proteins were immunoprecipitated
using anti-tag antibody, and the immunocomplexes were assayed for
histone deacetylase activity. Fig. 2B shows that both mHDA1
and mHDA2 possess histone deacetylase activity that is
TSA-sensitive.
The Expression of mHDA1 and mHDA2 in Various Mouse Adult
Tissues--
A Northern blot containing poly(A+) RNA isolated from
different mouse tissues was probed successively with mHDA1 and mHDA2 probes. These genes are expressed at various levels in different tissues but showed an interesting pattern of expression in the testis.
Indeed, in this tissue, mHDA2 is overexpressed (Fig.
3, mHDA2) and interestingly, a
truncated version of mHDA1, is present (Fig. 3, mHDA1,
Full-length probe). To know which part of the mHDA1-coding
sequence is missing in this testis-specific form, we designed a new
probe covering only the non-deacetylase domain. The probing of the
above Northern blot showed the same pattern of expression as the
full-length probe in all the tissues, except in testis, where the
truncated species was not detected (Fig. 3, mHDA1, -HD
probe). This result shows the expression of a testis-specific variant of mHDA1, which has conserved the deacetylase domain but lost
the large nondeacetylase domain. This testis-specific species of mHDA1
is most probably generated as a result of an alternative splicing that
removes the major part of the nondeacetylase region of the protein.
mHDA1 and mHDA2 Are Expressed in a
Differentiation-dependent Manner--
The observation of
the particular expression pattern of mHDA1 and mHDA2 in testis, a
tissue where the differentiation program is associated with drastic
chromatin remodeling, prompted us to examine the expression pattern of
these genes during cell differentiation in general. First, we used two
inducible in vitro differentiation systems. Murine
erythroleukemia cell (MEL cells) line is consisted of proerythroblasts
transformed with Friend virus complex and established in culture. The
differentiation of these cells can be induced using a variety of
chemicals such as hexamethylene bisacetamide (23). RNA was prepared
from cells taken at various times after the treatment with the inducer.
Interestingly, the expression of both mHDA1 and mHDA2 was strongly
induced 4 to 6 h after the addition of hexamethylene bisacetamide
(Fig. 4A). To confirm the
relationship between cell differentiation and mHDA1 and mHDA2
expression, we used another in vitro differentiation system,
that of a murine melanoma cell line, B16. The differentiation of these
cells was induced after the treatment of cells with 5 mM
butyrate, leading to the arrest of cell proliferation and the expression of melanin (19). Here again, the induction of cell differentiation was accompanied by an induction of both mHDA1 and mHDA2
expression (Fig. 4B). To exclude any artifactual expression of these genes in vitro, after the treatment with chemicals,
we tested an inducible in vivo system: rat liver after
partial hepatectomy. Indeed, partial hepatectomy induces liver cell
proliferation to regenerate the functional differentiated tissue (24).
Here, we also observed that mHDA1 and mHDA2 mRNAs accumulated at
24 h after partial hepatectomy (Fig. 4C). The
accumulation of these two messengers seems therefore tightly linked to
signals controlling cell differentiation, because the same pattern of
histone H1° mRNA accumulation was observed during this process
(22).
mHDA1 and mHDA2 Are Trichostatin A-responsive Genes--
We found
that the expression pattern of mHDA1 and mHDA2 in MEL cells, B16 cells,
and during liver regeneration is similar to the expression pattern of
histone H1° during these process (19, 22, 25). This observation
suggests a coordinate expression of H1° and these histone
deacetylases. To further investigate the possibility of such coordinate
expression, we tested the ability of mHDA1 and mHDA2 to be induced upon
histone deacetylase inhibitors treatment. Indeed, previous work using
various systems reported a strong induction of H1° gene expression
after treatment of the cells with histone deacetylase inhibitors (20,
26-28). The most convenient cellular system we used to show the
relationship between histone acetylation and H1° gene expression
consisted of two cell lines: FM3A and TR303 (20). The TR303 cell line
is a mouse mammary tumor cell line isolated from the FM3A parental cell
line after a general mutagenesis of cells and a selection procedure
based on the ability of individual clones to grow in the presence of TSA (29). The analysis of the level of histone acetylation showed that
in contrast to FM3A cells, the treatment of TR303 cells with low
concentrations of TSA does not lead to a hyperacetylation of core
histones (20, 29). These two cell lines provide therefore, an
interesting system to study the relationship between the general level
of histone acetylation and specific gene expression.
As expected, the treatment of FM3A cells with 5 and 20 ng/ml TSA for
6 h strongly induced the expression of mHDA1 and mHDA2 (Fig.
5A). In the TR303 cell line,
incapable of accumulating hyperacetylated histones under these
conditions of treatment, no accumulation of mHDA2 mRNA was
observed, and mHDA1 mRNA was slightly induced. Fig. 5B
confirmed the fact that the treatment of TR303 cells with these
concentrations of TSA for 6 h did not lead to the accumulation of
hyperacetylated histone H4 isoforms (the use of anti-hyperacetylated H4
antibody did not show the accumulation of these species). However, the
treatment of this cell line with these low concentrations of TSA did
lead to a major accumulation of monoacetylated histone H4 (see Fig.
5C and Refs. 20 and 29). In these cell lines, the expression
of glyceraldehyde-3-phosphate dehydrogenase, like many other genes,
remained unaffected by TSA treatment.
In this work we have identified a new family of higher eukaryote
histone deacetylase that is distinct from the previously defined HDAC
(RPD3) family. Indeed, in contrast to all animal deacetylases already
defined, their histone deacetylase homology domain is more related to
yHDA1 than to yRPD3. We therefore named these proteins mHDA1 and mHDA2,
members of a new histone deacetylase family in higher eukaryotes. mHDA1
and mHDA2 are able to deacetylate histones in vitro and
present sequence features, characteristic of histone deacetylases.
Indeed, most of the amino acids conserved between the RPD3/HDAC family
and prokaryotic acetylpolyamine amidohydrolase and the acetion
utilization protein, acuC (9-11), are also present in the deacetylase
homology domains of mHDA members (not shown). In particular, all of the
histidines and aspartic acids with potential catalytic and metal
coordinating properties (12), are absolutely conserved in mHDA1 and
mHDA2 (see Fig. 1), confirming the deacetylase nature of these members.
One of the deacetylases reported in this work, mHDA2, possesses two
deacetylase homology domains. This is the first enzyme of this type
reported to have this special feature. The significance of this
particular structure is not clear. mHDA1 possesses one deacetylase
homology domain located in the C-terminal part of the protein; the
N-terminal half of mHDA1 (over 500 amino acids) does not show sequence
homology to a known deacetylase domain nor is homologous to any known
proteins. This region is therefore a good candidate for regulating the
function of the protein and to mediate its interaction with potential
partners. The deletion of this nondeacetylase domain in the
testis-specific variant of mHDA1 supports this hypothesis. Indeed, this
large domain may not only regulate the enzymatic activity but may also target the protein to specific complexes. The deletion of this domain
could therefore be a way to obtain a more general and nonspecific deacetylase that is needed for the regulation of histone acetylation during spermatogenesis (30).
Different members of HDAC family have been found in different
regulatory complexes (1, 2, 31), and the identification of proteins
involved in these complexes was essential for the understanding of the
specific function of these deacetylases. The nature of these partners
helped to show that besides the involvement of HDAC family members in
chromatin assembly (32), the regulation of transcription is their major
function. Indeed, HDACs have been found in various complexes containing
different known repressors of transcription (1, 2, 31, 33, 34). These
enzymes also have the potential to sustain a chromatin-remodeling
activity. Nevertheless, this chromatin-remodeling activity is either
too modest to be visible by classical techniques (35) or operates locally at the level of nuclease hypersensitive sites (36). The
identification of cellular proteins able to interact with the mHDA
members is certainly crucial to understanding the specific function of
these proteins. The remarkable pattern of expression of mHDA1 and mHDA2
in testis (the overexpression of mHDA2 and the appearance of a
truncated form of HDA1, which has lost the nondeacetylase domain) is
the only evidence we have at present concerning their possible
function. Indeed, in this tissue, cell differentiation is associated
with spectacular chromatin remodeling, characterized by the replacement
of histones by transition proteins and protamines that could involve
histone hyperacetylation/deacetylation (30). Such a special pattern of
expression was not detected for members of the HDAC family in testis
(7). One can therefore suggest a specific involvement of the mHDA
family in chromatin remodeling during spermatogenesis and cell
differentiation in general.
mHDA1 and mHDA2 belong to a group of genes that show a
coordinate-induced expression in response to the inhibition of histone deacetylase activity. Interestingly, members of this group consist of
the histone deacetylases, mHDA1, mHDA2 (this work), HDAC1 (37), and a
differentiation-specific linker histone, H1° (20, 26-28). Moreover,
we observed that p21/WAF is also induced after the inhibition of
deacetylase activity by TSA (data not shown), as has been observed after butyrate treatment (38). Interestingly, p21/WAF has been shown to
play a major role as a nuclear organizer (39). The response of these
genes to chromatin hyperacetylation reveals the existence of an
interesting network of genes that are involved in the remodeling and
organization of chromatin, among them three histone
deacetylase-encoding genes, histone H1°, and the nuclear organizer,
p21/WAF. The fact that these genes are induced upon chromatin
hyperacetylation also indicates the existence of a chromatin-structure response system. The product of these genes could be involved in a
cellular response to severe disturbance of the regular chromatin structure. These genes also seem to be involved in other cellular events such as differentiation. The chromatin-modifying function of
H1° (21) and the histone deacetylase activity of the proteins identified here may play an important role in the correct remodeling of
chromatin during cell differentiation and the initiation of a
particular genetic program.
Our results strongly suggest that the mHDA1- and mHDA2-encoding gene
promoters like that of H1° gene, are communicating with cellular
signals controlling cell differentiation. It will therefore be very
interesting to identify transcription factors involved in the
coordinate expression of H1° and these histone deacetylases. The
identification of such factors would provide important insight into the
molecular nature of the chromatin-remodeling command mechanism
operating during cell differentiation.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
RESULTS
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Fig. 1.
mHDA1 and mHDA2 are members of a new mouse
histone deacetylase family. A, schematic representation
of distinct domains within mHDA proteins. The position of yHDA homology
domains are indicated (boxes). The black dots
indicate the position of the glutamate-rich (E-rich)
regions. B, the sequence of yHDA1 homologous domains of
mHDA2 (homology domain 1, HDD1, and homology domain 2, HDD2) and that
of mHDA1 were compared with the sequence of yHDA1 and yRPD3. The
identical amino acids present in yHDA1 and in mHDA1 and mHDA2 and yRPD3
are boxed. Numbers in the left indicate the
position (amino acids). Dots represent gaps, introduced for
alignment purposes. Asterisks indicate amino acids
identified by mutagenesis to play an important catalytic and/or
structural role in HDAC1 (12).
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Fig. 2.
mHDA1 and mHDA2 are histone
deacetylases. A, mHDA1 and mHDA2 are efficiently
translated in vitro and in vivo. mHDA1 and mHDA2
containing vectors were used to produce 35S-labeled
proteins in reticulocyte lysate after in vitro
transcription/translation (in vitro panel). Cells
transfected with the same vectors were lysed, and tagged mHDA1 and
mHDA2 were immunoprecipitated using anti-tag antibodies. A Western blot
was prepared to visualize the immunoprecipitated materials (in
vivo panel). B, after immunoprecipitation
using anti-tag antibodies, immunocomplexes were tested for histone
deacetylase activity. 100% (2000 cpm) represents HDAC1 activity used
as a positive control (mean value from two independent experiments and
two different measurements), and the bar indicates the range
of values obtained.
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Fig. 3.
Expression pattern of mHDA1 and mHDA2 in
different adult mouse tissues. A Northern blot containing 2 µg
of poly (A+) RNAs from indicated tissues was probed successively with
mHDA1, mHDA2, and actin probes (doublet in heart and testis is
because of probe hybridization to either the
or
form of actin).
The same blot was probed with a fragment of mHDA1 cDNA not
containing the deacetylase homology domain (-HD probe). Note
the disappearance of the small testis-specific transcript
(asterisks).
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Fig. 4.
The expression of mHDA1 and mHDA2 is tightly
linked to cell differentiation. A, murine
erythroleukemia cells were induced to differentiate with 4 mM hexamethylene bisacetamide for the indicated times, RNA
was prepared, and a Northern blot was obtained. The blot was probed
successively with mHDA1, mHDA2, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probes. B, murine melanoma
cells were induced to differentiate with 5 mM butyrate, and
RNA was prepared and analyzed as in A. C, rats
were partially hepatectomized, and the regeneration was allowed to
proceed for the indicated time. RNA was purified at the indicated times
and analyzed as above, except a 28 S rRNA probe was used as a
control.
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Fig. 5.
FM3A and TR303 cells were treated with the
indicated amount of TSA for 6 h, and the level of histone
acetylation and specific gene expression were analyzed.
A, RNA from FM3A and TR303 cells treated with different
amounts of TSA for 6 h was purified and analyzed. The Northern
blot was probed with the indicated probes. B, The use of
anti-hyperacetylated H4 antibody and flow cytofluorimetric analysis
allowed visualization of the accumulation of hyperacetylated form of
histone H4. Histograms represent the number of cells (y
axis) possessing a given immunofluorescence (H4 acetylation,
x axis). The concentration of TSA used to treat cell
populations is indicated above each histogram. C, the state
of histone H4 acetylation after the treatment of cells with 5 and 20 ng/ml TSA for 6 h was determined by electrophoresing total
histones on a Triton/acid/urea gel. The gel was stained with Coomassie
Blue.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jean Jacques Lawrence, the head of INSERM U309, for encouraging this work and to Dr. Stefan Dimitrov for critical reading of the manuscript, to Martina Creaven for English corrections, and to Vesco Mutskov, Marie-Paule Brocard, Sandrine Curtet, and Nathalie Bertacchi for technical assistance.
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FOOTNOTES |
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* This work was supported by Association pour la Recherche sur le Cancer (to S. K.).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) AF006602 (mHDA1) and AF006603 (mHDA2).
To whom correspondence should be addressed. Tel.: (33) 4 76 54 95 83; (33) 4 76 54 95 95; E-mail: khochbin{at}ujf-grenoble.fr.
The abbreviations used are: HAT, histone acetyltransferases; HD, histone deacetylases; TSA, trichostatin A; y-, yeast; m-, mouse.
2 http://dot.imgen.bcm.tmc.edu:9331/seq-search/nucleic_acid-search.html.
3 http://ujf-iab.ujf-grenoble.fr/IAB/cgibin/saadi/.
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REFERENCES |
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