Download The Modular Structure and Function of the Wheat HI Promoter with S

Plant Cell Physiol. 39(3): 294-306 (1998)
JSPP © 1998
The Modular Structure and Function of the Wheat HI Promoter with S
Phase-Specific Activity
Ken-ichiro Taoka 1 , Norihiro Ohtsubo 2 , Yoshinobu Fujimoto 3 , Koji Mikami 4 , Tetsuo Meshi1 and
Masaki Iwabuchi 1>5
1
Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-01 Japan
Two histone HI genes, TH31S and TH325, were isolated from a wheat genomic library. Nucleotide sequence analysis and comparison with other histone gene promoters
revealed that the promoters of both genes contain many
characteristic motifs conserved among plant histone HI
genes. They are 6 novel short stretches, named CS1 to CS6,
and already documented elements or their relatives such as
Oct, Oct-like (OLS), Nona-like (NonaLS), CCAAT box,
and TATA box. Transient expression experiments with the
TH315 promoter/GUS chimeric gene and its mutagenized
derivatives showed that two Oct motifs, OLS, and CCAAT
box are positive m-acting elements. NonaLS and CS4 were
suggested to be positive cw-acting elements and CSS and
CS6 to be negative elements. An Oct motif and CCAAT
box constitutes a type III element and the 202-bp sequence
containing these elements from —128 to +74 of the TH315
gene was shown to be sufficient to confer S phase-specific
expression. The type III element is found in all plant
histone HI and H2B genes, suggesting that it is a subtypespecific element. Most plant histone genes have one of the
type I, II, and III elements. We propose to classify the
plant histone genes into three classes, based on the context
of Oct in the promoters.
linker histone HI, which is thought to be associated with
DNA outside nucleosomes, sometimes resulting in transcriptional repression (Cronston et al. 1991, Laybourn et
al. 1991). Thus, core and linker histones play different roles
in chromatin condensation.
Gene expression of the two classes of histones is controlled in a coordinated and S phase-specific manner (for
review, Osley 1991, Stein et al. 1994). Control of such
histone gene expression is exerted at multiple levels: transcription, 3'-processing of pre-mRNA, and mRNA stability
(for review, Osley 1991, Stein et al. 1994). Among them,
transcriptional regulation has been extensively studied. In
vertebrate histone genes, the cis-acting elements involved in
the S phase-specific transcription have been identified, and
it is suggested that the coordinated expression of histone
genes is regulated in a subtype-specific manner (for review,
Osley 1991, Stein et al. 1994). In yeast histone genes, the
cis-acting elements commonly existing in all the core
histone genes are involved in S phase-specific transcription
(for review, Osley 1991).
In plant histone genes, five cis-acting elements, Hex
(CCACGTCA), Oct (CGCGGATC), Nona (CATCCAACG), ACT box (CACTC), and the CCGTC motif have
been identified for the H3 and H4 genes of wheat,
maize and Arabidopsis (Chaubet et al. 1996, Lepetit et al.
1993, Nakayama et al. 1992, Sakamoto et al. 1995, Terada
et al. 1995). The Oct motif is found in all the plant histone
gene promoters known to date. The cis-acting region containing the Oct motif has been recognized as a type I, II or
III element, based on the difference in the flanking sequences
and the presence of other cis-acting sequences (Mikami and
Iwabuchi 1993, Yang et al. 1995). The type I element
(CCACGTCANCGATCCGCG) is composed of the Hex
motif and the reverse-oriented Oct motif. This composite
element is known to regulate S phase-specific gene expression (Ohtsubo et al. 1997) and to be involved in
meristematic tissue-specific gene expression (Terada et al.
1995). The type II element (TCACGCGGATC), an 11-nucleotide sequence including the authentic Oct motif as the
core, is found in the promoter regions of some plant
histone genes that can direct replication dependent- and
meristematic tissue-specific expression (Atanassova et al.
1992, Chaubet et al. 1996, Huh et al. 1997, Lepetit et al.
1992). The type HI element (CGATCCGCGN14ACCAATCA) is composed of the reverse-oriented Oct and the CCA-
Key words: cis-Acting elements — Histone HI gene — Promoter analysis — S phase-specific expression — Triticum
aestivum.
Histone proteins are classified into two distinct classes
in terms of function. One class consists of four subtypes,
H2A, H2B, H3, and H4, called core histone proteins.
Their octameric structure forms a nucleosome core particle
to package chromosomal DNA. The second class is the
The nucleotide sequences reported in this paper have been
submitted to DDBJ, EMBL and GenBank Databases under accession numbers D87064 (TH315) and D87065 (TH325).
2
Present address: National Institute of Agrobiological Resources, Tsukuba, 305 Japan.
3
Present address: Nippon Oil Company Ltd., Kudamatsu, 744
Japan.
4
Present address: Department of Regulation Biology, National
Institute for Basic Biology, Okazaki, 444 Japan.
5
Author for correspondence.
294
Promoter analysis of a wheat HI gene
295
cleotide-aided method (Kunkel et al. 1991) with ssDNA from
pSUB128TH315, which was constructed by inserting the Pstl/
Smal fragment of —128 HI/GUS into the corresponding sites of
pBluescript II KS (—). Synthetic oligonucleotides used were:
mGC, 5-CAACTAGGGATGGCTTGATT-3'; mOct-d, 5'-GGGCTTGAAGACGCGGCATC-3'; mOLS, 5'-ATCCGCGGACGAGGATGGGG-3'; mOct-p, 5-GGGGGGCGCGAAGTGGCAAC-3'; mCCAAT, 5'-CCCTTGACCCATCAGCGCGC-3'.
All the chimeric gene constructs mentioned above were checked by sequencing them.
RT-PCR analysis—RT-PCR analysis was performed as described previously (Jayawardene and Riggs 1994). Two-hundred
ng of the total RNA from wheat seedlings was digested with
RNase-free DNase I to remove a trace amount of the contaminated genomic DNA. cDNA was synthesized using the Superscript preamplification system (GIBCO BRL) according to the
manufacturer's instructions. The cDNA derived from 200 ng of
the total RNA, 1 fig of wheat genomic DNA, and 500 pg of
pTH315 were used as templates for PCR in 50^il of the reaction
mixture containing the primers termed 315UP, 5-AGGCCAAGGCCGCCAAGACC-3', corresponding to the sequence from
+ 204 to +223 of TH315 and 315LOW, 5-TTCCTCTCCTTCAGCGCGGC-3', corresponding to the complementary sequence from +460 to +441 of TH315, and ExTaq DNA polymerase (TaKaRa Syuzo). Amplification was 35 cycles at 95°C for
60 s and at 65°C for 60 s. Amplified DNAs were electrophoresed
on 5% acrylamide gels and visualized by staining with ethidium
bromide. PCR primers designed were specific to TH315 and no
amplified product was detected when the cloned TH325 gene was
Materials and Methods
used as a template.
Transient expression assay in cultured rice cells—Preparation
Isolation of wheat HI genes—A wheat genomic DNA library
of
protoplasts
from suspension-cultured rice cells (cv. Yamawas screened as described previously (Mikami et al. 1995) using
houshi), electroporation of plasmid DNAs and fluorometric measwcHl-1, a wheat HI cDNA (Yang et al. 1991), as a probe. Two
positive clones were obtained and designated A315 and A325. The urement of GUS activity were carried out as described previously
(Ito et al. 1995).
EcoRl fragments containing the histone HI gene were subcloned
Transformation and synchronization of cultured rice cells,
into the EcoRl site of pUC118 to generate pTH315 and pTH325.
fHJthymidine pulse-labeling, and Sl-nuclease protection assay—
Overlapping deletions of the inserts were generated in both direcTransformation of cultured rice cells, synchronization of the cell
tions as described previously (Henikoff 1984) and nucleotide secycle, [3H]thymidine pulse-labeling, and Sl-nuclease protection
quences were determined by the dideoxy chain termination
assay were carried out as described previously (Ohtsubo et al.
method (Sanger et al. 1977).
1993). Total RNAs were prepared from transformed rice cells usPlasmid construction—A Haelll fragment of pTH315 (from
ing
the TRIzol reagent (Life Technologies, Inc.) according to the
— 63 to +198, see Fig. 1A) was subcloned into the Smal site
manufacturer's instructions. The radioactivity of Sl-nuclease
of pUC118 to generate pTH315HH. The HI promoter/GUS
chimeric gene — 771H1/GUS was constructed by inserting an digested products was measured with a Bio-Imaging Analyzer
(Fuji Film).
blunt-ended EcoRl/Sphl fragment of pTH315 (from - 7 7 1 to
+ 74) into the blunt-ended BamHl site of pNGN, located between
the upstream nos terminator and the promoter-less GVS/nos terResults
minator fusion gene (Ohtsubo et al. 1997). pDELTH315 was constructed by inserting a Pstl/Smal fragment of -771H1/GUS
Cloning and characterization of wheat histone HI
(from —771 to +74) into the corresponding sites of pBluescript II genes—Two wheat histone HI genes were isolated from a
KS ( - ) . pDELTH315 was digested with Pstl and Xbal, deleted,
genomic library with the wheat HI cDNA wcHl-1 (Yang et
then self-ligated. The resulting plasmids retaining the sequence
al.
1991) as a probe. Restriction mapping and sequencing
from - 5 9 0 to +74 and that from - 4 0 1 to +74 of TH315 were
revealed that the isolated genomic clones contained distinct
digested by Kpnl, blunted by the Klenow fragment, and digested
by BamHl to recover the HI promoter fragments. The recovered
but closely related HI genes (Fig. 1). The nucleotide sefragments were recloned between the blunt-ended Xbal and
quences of these genes, named TH315 and TH325, were
BamHl sites of pNGN to generate -590H1/GUS and - 4 0 1 H 1 /
very similar (about 90% identity in the sequenced regions).
GUS. The other 5' deletion constructs were prepared from
-401H1/GUS by unidirectional deletion from the Xhol site. The Neither of the two genes corresponds to the cDNA used as
the probe; the identity was about 80% and 60% at the nuresulting mutant constructs with a 5' truncated promoter were
named —xxHl/GUS, where —xx means the 5'end position of the cleotide and deduced amino acid sequence levels, respectiveshortened promoter.
ly.
Comparison of the genomic and cDNA sequences sugBase-substitution mutations were introduced by the oligonu-
AT box separated by 15 bp. This composite element is conserved among all the plant HI and H2B genes known to
date, and the wheat H2B promoter containing the type III
element can direct meristematic tissue-specific expression
(Yang et al. 1995).
The regulatory mechanisms of the genes coding for the
core and linker histones apparently differ. In addition to
normal S phase-specific expression, there have been reports
which indicate that HI mRNA levels are high in tissues
with less proliferating activity (Minami et al. 1993, Razafimahatratra et al. 1991), and a tobacco HI mRNA is
regulated diurnally (Szekeres et al. 1995). To begin to explore plant HI gene expression, we cloned two wheat HI
genes. A comparison of the sequence data showed that the
promoter region of these HI genes has a modular structure
composed of several short stretches conserved among plant
HI genes. Mutational analyses of the promoter region of
the wheat HI gene TH315 revealed that two Oct motifs,
OLS, and CCAAT box are positive as-acting elements and
that the 202-bp region containing the type III element can
confer S phase-specific expression. We suggest an important role of the type III element in the S phase-specific regulation of plant HI genes.
296
Promoter analysis of a wheat HI gene
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Fig. 1 Nucleotide and deduced amino acid sequences of wheat HI genes, TH315 (A) and TH325 (B). The deduced amino acid sequences are shown in single capital letters below the nucleotide sequences. The asterisks denote the stop codons. The transcriptional initiation site (+1), the deletion points described in the text, and the coding sequences are represented by bold letters. Introns are written
in lower case letters. The characteristic sequences described in the text are underlined.
gested the existence of an intron in the protein-coding
region. The putative introns are bracketed by consensus
splice site dinucleotides. To confirm this, PCR analysis was
performed with the first strand of cDNA and nuclear DNA
as templates (Fig. 2). When the primers hybridizing to the
sequences upstream and downstream of the putative intron
were used, the same size (257 bp) of the DNA fragment was
amplified from both the cloned TH315 gene and the nucle-
Promoter analysis of a wheat HI gene
A
31 SUP D — ^
89bp
genomic DNA
•*—D315LOW
257bp
315UPD-*"
89bp
38bp
cDNA
127bp
-^-Q315LOW
B
1 2
3
4
5
Fig. 2 Identification of an intron in the wheat HI gene TH315
by RT-PCR analysis. A, Schematic representation of predicted
PCR products from the genomic DNA and cDNA when two
primers (315UP and 315LOW) are used. Open boxes represent
part of exons 1 and 2, and the closed box represents the intron. B,
Polyacrylamide gel electrophoresis of amplified products. Lane 1,
ADNA digested with EcoT14l; lane 2, 100 bp ladder; lane 3, amplified product from the cloned genomic fragment containing the
wheat HI gene TH315; lane 4, amplified product from the wheat
genomic DNA; lane 5, amplified product from cDNA prepared
from the total RNA extracted from wheat seedlings.
ar DNA, whereas the fragment amplified from the cDNA
was shorter, corresponding to the expected size of 127 bp.
Nucleotide sequencing confirmed that the amplified product was from the TH315-derived transcripts (data not
shown). Thus, it was concluded that the TH315 gene has an
intron and is expressed in vivo. The transcription initiation
site was determined by the SI nuclease mapping and primer
extension methods (data not shown).
Characteristics of the HI proteins encoded by TH315
and TH325—Histone HI proteins encoded by TH315 and
TH325 are composed of 284 and 288 amino acids, respectively, with three characteristic domains conserved in all
HI histones: Lys-rich amino terminal domain, hydro-
297
phobic 'central globular domain', and Lys-rich carboxy terminal domain.
Amino acid sequence comparisons of the wheat
TH315 protein with other plant His showed that similarity was rather restricted to the central globular domain
(Fig. 3A), ranging from 80% for wcHl-1 (Yang et al. 1991)
to 50% for Arabidopsis Hisl-3 (Ascenzi and Gantt 1997).
In a phylogenetic tree, plant HI proteins can be classified
into at least three subgroups (Fig. 3B). The first group is HI
proteins from dicotyledonous plants. The second group is
monocotyledonous HI proteins including the wheat proteins encoded by TH315 and TH325. The third group contains minor variants that are considerably divergent from
the former two, including variants of tomato and Arabidopsis that are induced in response to drought and ABA
(Ascenzi and Gantt 1997, Wei and O'Connell 1996). Hereafter, the first and second groups are referred to as major
types to contrast them with the third group.
Characteristic sequences in the 5' upstream regions of
TH315 and TH325—Several characteristic sequences were
found in the promoter regions of the TH315 and TH325
genes (Fig. 1). The Oct motif is conserved in almost all the
plant histone gene promoters known to date, and it has
been shown to be a positive cis-acting element in some
plant H3 and H4 genes (Chaubet et al. 1996, Nakayama et
al. 1992, Terada et al. 1995). Two Oct motifs were present
in both TH315 and TH325 genes, and they were named
Oct-p (proximal) and Oct-d (distal).
The Oct-d sequence overlaps an Oct-like sequence
(OLS, CGCGGCATC), and the Oct-p sequence forms a
type III element in combination with a CCAAT box, in
which the two sequences are located with a 15-bp interval.
Other characteristic motifs found were a Nona-like sequence (NonaLS, GATCGGACG), a GC box (GGGCGGG), and an AC box-like sequence (AAACCCAAATCACACAAA). The Nona sequence is known to be a positive
m-acting element in some plant H3 and H4 genes (Chaubet
et al. 1996, Lepetit et al. 1993, Nakayama et al. 1992). The
GC box and AC box are highly conserved among vertebrate HI genes and their involvement in gene regulation
has been demonstrated (for review, Osley 1991).
Comparison of the sequences of histone HI promoters
of wheat, Arabidopsis and tomato revealed six kinds of
conserved stretches (named CS1 to CS6), in addition to the
motifs mentioned above (Fig.4A). Interestingly, the positional relationship between these conserved stretches and
some of the above-described motifs (NonaLS, Oct-p and
CCAAT box) is also conserved (Fig. 4B). CS5 and CS6 include a pentameric motif, CCGTC, as the core, which has
been suggested to be a proliferation-induced binding site
for a transcription factor(s) in the maize H3 and H4 promoters (Brignon and Chaubet 1993) and has been shown to
be a cis-acting element for tissue-specific expression of the
Arabidopsis H4 genes (Chaubet et al. 1996). The four other
298
Promoter analysis of a wheat HI gene
T
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B
• Tobacco(H1c12)
• Tomato (U03391)
- Arabidopsis (H1-1)
- Arabidopsis (H1-2)
- Pea (Hib)
- Pea(H1-41)
• Wheat (TH325)
• Wheat (wcH1-1)
- Wheat(TH315)
• Maize (CH1C21)
Tomato (U01890)
Tomato (Z11842)
Arabidopsis (His1-3)
Fig. 3 A comparison of the primary structure of plant HI proteins. A, An alignment of the amino acid sequence of the central
globular domain of plant HI proteins known to date. Gaps introduced to maximize the alignment are indicated by dashes. Asterisks
mean the amino acid residues identical to that of the TH315 protein. B, Phylogenetic tree of plant HI proteins, drawn according to the
UPGMA method. The values on the tree mean the relative distance to the former branching point. Sequence data are: wheat (this paper,
Yang et al. 1991), maize (Razafimahatratra et al. 1991), Arabidopsis (Ascenzi and Gantt 1997, Gantt and Lenvik 1991), pea (Gantt and
Key 1987, Woo et al. 1995), tobacco (Szekeres et al. 1995) and tomato (Jayawardene and Riggs 1994, Wei and O'Connell 1996).
conserved stretches, CS1 to CS4, have not been recognized
as cis-acting elements. It should be noted that these conserved stretches are not found in the genes for minor
variants such as the drought-inducible HI genes of Arabidopsis and tomato.
Functional analysis of the promoter of TH315—To
delineate functional regions of the TH315 promoter, a
series of 5' deletion mutants in which one or two characteristic motifs had been removed were constructed, and their
promoter activities were examined using the transient assay
299
Promoter analysis of a wheat HI gene
CS1
Wheat(TH315)
CS2
TCCACAAGT- -TGTGCAAC
ATCTCATTACAAA
CS3
CTACACGATGAAAAAAA
Wheat (TH325)
TCCACAACT- -TGTGCAAC
ATCTCATTACAAA
CTACACGATGGAAAAAA
Arabidopsis (H1-1)
TCAACAAGTAGTGTGCAAC
ATCTCATTACAAA
CTACACAATGCAAAAAA
CTACACGGTGAAAAAAA
Tomato (U03391)
CS4
CS5
Wheat (TH315)
AATCGAGAGGAGAAGCACA
GCCGTCGGAT
Wheat (TH325)
AATCGAGAGGAGAAGCACA
GCCGTCGGAT
Arabidopsis (H1-1)
AATCGAGTCTAAAAGCACA
GCCGTC-GAT
Tomato (U03391)
AATTGAGCATAGAAGCACA
ACCGTT-GAT
B
-600
-400
/
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-200
i
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. /
Wheat (TH315)
(TH32S)
Arabidopsis
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Tomato (Hist)
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Arabidopsis (Hisi-3)
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Fig. 4 Conserved sequences found in the promoter regions of plant HI genes. A, Conserved stretches designated as CSl to CS6 are
aligned. The CCGTC sequences found in CS5 and CS6 as the core are indicated by brackets. The drought-inducible HI genes (Ascenzi
and Gantt 1997, Wei and O'Connell 1996) have no CS motifs. It is unknown whether the tomato HI gene (U03391) has CSl and CS2, because the nucleotide sequence of the corresponding region is not available. B, Schematic representation of the positions of characteristic
motifs in the plant HI promoters. AC box, AC box-like, GC box, Oct-d, OLS, Nona, NonaLS, Oct-p, CCAAT box and TATA box, see
text. Note that the locations of Nona/NonaLS, Oct-p, CCAAT box, TATA box, and CSl to CS5 are highly conserved in the plant HI
genes, with the exceptions of the drought-inducible HI genes (Ascenzi and Gantt 1997, Wei and O'Connell 1996). The scale at the top indicates the position from the transcription initiation site.
system in protoplasts from suspension-cultured rice cells
(Fig. 5). GUS activity of the chimeric gene with the promoter sequence up to - 7 7 1 ( —771H1/GUS, the full-sized con-
struct) was set to 100%.
The GUS activity of -771H1/GUS was comparable
to that of -185H3/GUS, a wheat H3 promoter-GUS
300
Promoter analysis of a wheat HI gene
-771M1/GUS
-45H1/GUS
-28H1/GUS
50
100
150
200
Relative GUS activities (%)
Fig. 5 Transient expression analysis of a series of 5' deletion mutants of the —771H1/GUS chimeric genes in rice (cv. Yamahoushi)
protoplasts. The protoplasts were transfected with chimeric constructs. Relative GUS activities are indicated by the average values of duplicate assays for four independent preparations of protoplasts (eight assays for one construct) with the value for — 771H1/GUS as
100%. T-bars represent standard errors.
chimeric gene (Ito et al. 1995). Large decreases in the promoter activity were observed when the regions from —314
to - 2 5 0 , from - 2 4 9 to - 1 9 6 , from - 1 5 3 to - 1 2 9 , and
from —45 to —29 were deleted. The motifs contained
in these deleted regions are CS4, GC box plus Oct-d,
NonaLS, and TATA box, respectively, and accordingly,
they are candidates for positive c«-acting elements. Slight
but significant decreases were also observed by deleting the
regions from - 7 7 1 to - 5 9 1 , from —128 to - 9 7 , and
from —96 to —67, which contain motifs, CS1, Oct-p, and
CCAAT box, respectively. On the other hand, a rise in promoter activity was observed by deleting the regions from
- 5 9 0 to - 4 0 2 , from -181 to - 1 5 4 , and from - 6 6 to
— 46, which contain the CS5, and CS6 sequences.
The Oct motif is often paired with another motif and
in the case of the type I element, cooperation with Hex has
been reported (Ohtsubo et al. 1997). In the TH315 and
TH325 genes, Oct-p is a part of the type III element and
Oct-d overlaps with OLS (Fig. 6A). In addition, a GC box
can be found just upstream of Oct-d, just at the position
corresponding to Hex in the type I element. To clarify the
involvement of the Oct-p and -d motifs in the promoter
function and to examine their cooperation with adjacently
located motifs, base-substitution mutations were introduced into the -771H1/GUS and -128H1/GUS genes and
the mutant promoter activities were measured. As shown in
Fig. 6B, the mutations in Oct-d and -p, OLS, and CCAAT
box significantly decreased the promoter activity, consistent with the results of the deletion mutants, whereas the
mutation in the GC box had little effect on promoter activity. This suggests that Oct-d is the major element in the
decreased promoter activity caused by a simultaneous
deletion of GC box and Oct-d (see -249H1/GUS and
-196H1/GUS in Fig. 5). Since similar effects were observed between Oct-d and OLS mutations (Fig. 6B) and between deletions of Oct-d and of both Oct-d and OLS
(Fig. 5), these two overlapping elements might function as
a whole.
S phase-specific expression of the HI gene (TH315)—
The fact that the TH315-encoding protein belongs to the
major type of histone HI implies that its expression may
be regulated in a cell cycle-dependent manner. To know
whether the promoter region of the wheat HI gene TH315
is able to confer S phase-specific expression, a rice cell
line was transformed with the -128H1/GUS and Adhl
promoter/hygromycin phosphotransferase (hph) chimeric
genes. Two independently transformed lines, 128-2 and
128-7, were analyzed in detail, and essentially the same
results were obtained (Fig. 7).
The cell cycle of the transformed cells was partially synchronized by the aphidicolin method as described previously (Ohtsubo et al. 1993); DNA synthesis is blocked and progression of the cell cycle is arrested at the Gl/S boundary
in the presence of aphidicolin, and upon removal of the
drug, they resume. At 1 h after removal of the drug, the
rate of DNA synthesis reached the maximum and then
rapidly decreased (Fig. 7). The amount of mRNA from the
— 128H1/GUS gene also reached the maximum at 1 h after
301
Promoter analysis of a wheat HI gene
A
-771
v
GC box
Oct-d
• GGGGSfcGCTTGAAEEGCGGEglBl •
Oct-p
CCAAT box
•GEEJ33TG-
• -ACCSVTCAG •
OLS
AT
mGC
GA
ACGA
mOct-d mOLS
CGAA
mOct-p
c
mCCAAT
B
-771H1/GUS
-771HlmGC
-771HlmOct-d
-771HlmOLS
-771HlmOct-p
-771HlmCCAAT
-771HlmGC/mOct-d
-771HlmGC/mOLS
-771HlmOct-d/mOct-p
-771HlmOct-p/mCCAAT
-128H1
-128HlmOct-p
-128HlraCCAAT
-128HlmOct-p/mCCAAT
Relative GUS activities (%)
Fig. 6 Transient expression analysis of base-substitution mutants of the Hl/GUS chimeric genes in rice protoplasts. A, Base substitutions introduced into the HI promoter. The shadowed bases were substituted to the bases shown below with the names of the respective
mutations. B, Transient promoter activities of the base-substitution mutants of the —771 and -128H1/GUS chimeric genes in rice
protoplasts. Relative GUS activities are shown as in Fig. 5.
release of aphidicolin, then decreased rapidly. This pattern
conforms to the pattern of DNA synthesis, although the
amount of the Hl/GUS mRNA at 0 h was large, compared
with the patterns for H3 and H2A promoters (Huh et al.
1997, Ohtsubo et al. 1993, 1997). On the other hand, the
amount of the Adhl/hph mRNA used as an internal
reference did not appear to change during the cell cycle
(Fig. 7). The rRNA amount was not significantly changed
(data not shown). Such an S phase-specific accumulation of
the Hl/GUS mRNA was also observed with tobacco BY-2
cells transformed with the — 128H1/GUS gene (data not
shown). Therefore, these results suggest that at least one
e/s-acting element involved in S phase-specific expression is
located within the 202-bp region spanning from —128 to
+ 74 of the HI gene TH315.
Discussion
In the present study, we have isolated two distinct
wheat histone HI genes with an intron in the coding region
(Fig. 1, 2). In animals, the intron-containing histone genes
are known to be regulated in a cell cycle-independent man-
Promoter analysis of a wheat HI gene
302
B
128-2
128-7
I!
il
0
1
2
3
<
5
6
1
7
Time after release fam apHdcoin (h)
Adhilhph'
H1/GUS
2
3
4
5
6
Time after release from aptttfcoln (h)
Adh1lhpht>
t>
• — - ••
H1/GUS >
Fig. 7 S phase-specific expression of the GUS mRNA in transformed rice Oc cells with the - 128H1/GUS chimeric gene. The transformed cell lines 128-2 (A) and 128-7 (B) were independently prepared. Transformed cells were treated with aphidicolin (10 fig ml"1) for
24 h. After removal of the drug, DNA synthesis resumed and cells progressed into the S phase. Total RNA was extracted from transformed rice cells at 1 h intervals after release from aphidicolin and the amount of the HI/GUS mRNA was quantified by the SI nuclease protection assay. The relative amount of the mRNA was plotted with the amount at 0 h as 1.0. The amounts of the hph mRNA derived from
the maize Adhl promoter/hph chimeric gene, which had been simultaneously integrated, was concurrently assayed as a cell cycle-independent internal control (Ohtsubo et al. 1997). The SI nuclease-protected patterns for the Hl/GUS and Adhl/hph mRNAs are shown
below the graphs. Radioactivities were quantified with a Bio-Imaging Analyzer. DNA synthesis was monitored by [3H]thymidine pulse-labeling.
ner (for review, Osley 1991). Similarly, Arabidopsis genes
for the minor histone variant H3.3, containing two introns,
have been shown to be expressed at significantly high levels
in tissues and organs with low proliferating activity
(Chaubet et al. 1992, Kanazin et al. 1996). In contrast, all
the plant histone HI genes cloned so far have an intron at
the same position in the coding region, which roughly corresponds to the junction between the amino-terminal domain
and the central globular domain of the protein. Introns
have also been found in all the plant histone H2A genes
cloned (from wheat and Norway spruce) (Huh et al. 1997,
Sundas et al. 1993). Since the wheat HI and H2A promoters can confer S phase-specific gene expression (Fig. 7 and
Huh et al. 1997), the presence of intron is not necessarily related to cell cycle-dependency of histone gene expression in
plants.
Several characteristic motifs were found in the promoters of TH315 and TH325; they are positive ciy-acting
elements identified in plant (Oct, Nona, CCGTC) and
animal (AC box, GC box, CCAAT box) histone genes, and
novel motifs (CS1 to CS6) conserved in the promoter
regions of plant histone HI genes. It should be noted that
the relative positions of CS1 to CS5, Nona or NonaLS, Oct
(Oct-p in TH315), and CCAAT box are conserved in the
promoters of histone HI genes of wheat, tomato, and
Arabidopsis, which encode the major type of histone HI
proteins. This finding suggests that these motifs may func-
tion as cis-acting elements involved in regulation of the
genes for the major type of angiosperm histone HI. In contrast, these motifs except for the AC box, Oct, and
CCAAT box, were not found in the regulatory region of
the HI genes, coding for drought- and ABA-inducible
minor variants (Ascenzi and Gantt 1997, Wei and O'Connell 1996).
Deletion analyses of the TH315 promoter showed that
the region containing the TATA element was absolutely
necessary for expression (Fig. 5). Further, CS4 and NonaLS are probable candidates for the ciy-acting elements
to explain large decreases in the promoter activity by 5' deletions (Fig. 5). Base-substitution mutations confirmed
that Oct-p, Oct-d, CCAAT box, and OLS are positive
cis-acting elements (Fig. 6), and considering the effects of
deletions, Oct-d and OLS were suggested to act together as
one element.
On the other hand, deletions of the sequence from
—181 to -154 and from —66 to —46 resulted in increases
in the promoter activity (Fig. 5). These regions therefore appeared to have negative elements and they may be CS5 and
CS6. Functions of other conserved motifs, CS1, CS2, and
CS3, could not be clarified, because the effects of their deletions were small. These motifs may be involved in tissuespecific expression of histone HI genes or other regulation
systems that can not be elucidated by the transient assay. In
relation to this, diurnal fluctuation of the gene encoding a
Promoter analysis of a wheat HI gene
tobacco HI protein has been observed (Szekeres et al.
1995).
Of the two Oct motifs in the TH315 and TH325 promoters, Oct-p forms the type III element in combination
with the CCAAT box. The type III element was first recognized in the promoter regions of wheat H2B genes (Yang et
al. 1995). Extensive search for the type III element in plant
histone promoters revealed that this element is present in
all plant HI and H2B promoters, and a few H3 promoters
(Fig. 8). In addition, the following common features were
found (Fig. 8): (1) the distance between the Oct and
CCAAT motifs (15 bp in principle), (2) conservation of the
sequence around the CCAAT box (ACCAATCA), and (3)
the distance from the CCAAT box to the TATA box
(about 40bp for HI genes and 20bp for H2B genes).
Since the type III element is always found in the HI
H1
Oct
Wheat (TH315)
Wheat (TH325)
Arabidopsis
(AtH1-1)
14 bp
303
and H2B gene subtypes, this element seems to be crucial
for the subtype-specific expression of plant histone genes
(Table 1). Although mutational analyses showed that Oct-p
and CCAAT box function as positive cis-acting elements,
their synergestic action could not be detected. The function
of Oct-p seemed dependent on the presence of CCAAT box
in the context of the — 771H1 promoter, but the effect of
the two elements were rather additive in the — 128H1 promoter (Fig. 6). In the case of the type I element, cooperation of the Hex and Oct motifs is clearly evident in S phasespecific expression (Ohtsubo et al. 1997), which cannot be
revealed by the transient assay system with protoplasts.
Considering that the — 128H1 promoter, which contains a
type HI element and CS6, is sufficient for conferring S
phase-specific expression in transformed rice cells, Oct and
CCAAT box in the type III element may function in an in-
CCAAT box
CS6
TATA box
TAGaXSGGGCOATCCOTOCXMCGCTCCCTTCyvCCAATCAGCGCGCGGCATT^^
TTATGGGGGCOATCCeXSKCAATGCCCTCTCGMX^TCA^
AAAGAGTAGCOATCCOCOTTIGTCATICTTTTAACCJUITCAGAAGCCAM^
Tomato (U03391)
H2B
Oct
14 Dp
CCAAT box
TATA box
Wheat (TH123)
CCACCTCAACOMCOTIlACCCCAGCCCTCTCCACaVXTCACGCCCTrCGCGCAroCCTATATATTCCTAGC
Wheat (TH1S3)
ATATCATATCGMCCOIWX^CCCATCCATCAACCAATCA<»GCCCGCCTCCCTCCTCG*ATAAAA0CTGC
Maize (gH2B4)
CCATCTCAGC<»TCaXXKCGCCACCCXrrTCCACCA*TCACATCCCGCCTCACCTTTCT»TATAATCCCAG
type III element (consensus)
CGATCCGCG
(N)H
ACCAATCA
B
H3
Oct
^
Rice
(H3R-21)
CCAAT box
^
195 bp
GAAGCGATGCQATCCOCOCGCTTCCGCATCGACCAATCACAGCGCAG
^
14 bo
^
t
155 bp
(H3.I)
TTATTTCAGCaATCCOCaACGGTrrcTATTCAGCCAATAGCAATCAA
^
13 DO
,
„
124 Dp
(msH3g1)
CCACGCTGGCAATCCQCAACTTTACAAACCAACX^ATCAGAAACAAA
Arabidopsis
Alfalfa
13DP
drought-induclble H 1
Oct
CCAAT box
Tomato (His1)
AGAAAACTTO3AT«:WWICACCTTAAATTGACCAATCAAAATCCACAGAGTTGT^
,
13 DP
,
Tomato (LE20)
AGAAAACTTCGATCCOTtnCACCTTAAATTGACCAATCAAAAraXGAGAATTCTGA^
^
I3bp
,
Arabidopsis (Hisi-3)
TATA box
..
CCCTCTCCCCTATATAAACGCTGC
^
CGTCCGATCATATAAATCCGCTTT
^
AACCCTTGCATATATAAACACACT
TATA box
ACACGTAATa»TCCTCTGACAAAAACCATAACOAAIACAGAAAACACACGAATA(»CTrCCCTGCGCTATAAATAAGCTAGCACGA^
Fig. 8 Conservation of the type III element in the promoter regions of plant histone gene subtypes. A, An alignment of promoter
regions of plant histone genes containing a type III element. The type III element is highly conserved in the plant HI and H2B promoters. Note that the space between Oct and CCAAT box is strictly conserved, that the flanking sequences of Oct or CCAAT box are also
conserved, and that this element is located near the TATA box. B, The combinations of Oct and CCAAT box found in some other plant
histone promoters. Note that they are close relatives to the type III element but do not have all of the characteristics described above,
wheat H2B (Yang et al. 1995), maize H2B (Joanin et al. 1994), rice H3 (Wu et al. 1989), Arabidopsis H3 (Chaubet et al. 1992), alfalfa H3
(accession no. U09458). For others, see the legend of Fig. 3.
304
Promoter analysis of a wheat HI gene
Table 1 Classification of the plant histone genes based on the contexts of the positioning mode of the Oct motif
Gene type"
Histone
subtype
Type I gene
H2B
H3
TH153
TH012
PcH3-7, PcH3-16, PcH3-20
Y14195
ALH3-1.1
THOU
H4C7, H4C13
Wheat
Arabidopsis
Rice
Maize
Arabidopsis
Maize
TH224, TH254, TH274
H3.3-II, H3A725
H3R21
H3C2, H3C3, H3C4
H4A748, H4A777
H4C14
Wheat
Arabidopsis
Tomato
Wheat
Maize
TH315, TH325
AtHl-1
U03391
TH123, TH153
gH2B4
Tomato
Arabidopsis
Arabidopsis
Rice
Alfalfa
Hisl, LE20
His 1-3
H3.3-I
H3R21
msH3gl
H4
Arabidopsis
Rice
Wheat
H3A713
H3R11
TH091
H3
Alfalfa
msH3g423
H2A
H3
H4
Type III gene
HI
H2B
Type III gene relative*
HI
H3
Unclassifiablec
No Oct
Gene name or accession number
Wheat
Wheat
Parsley
Tobacco
Alfalfa
Wheat
Maize
H4
Type II gene
Plant species
H3
° Definition of each type is described in the text.
* Genes that have a type HI element, which is slightly different from the canonical type III element in the HI and H2B promoters (for
details, see Fig. 8).
c
Genes that have none of the type I, II, nor III elements but contain an Oct motif.
Tobacco H3 gene (accession no. Y14195), Wheat H2A genes (Huh et al. 1997), Arabidopsis H3.3-II (Chaubet et al. 1992), Arabidopsis
Hisl-3 (Ascenzi and Gantt 1997), alfalfa H3 msH3g423 (accession no. U09459). For type III genes and type III gene relatives, see
references in Fig. 8. For others, see references in Mikami and Iwabuchi (1993), and Takase and Iwabuchi (1993).
tegrated manner in cell cycle-dependent transcriptional regulation. We are currently investigating this possibility.
The Oct motif has been found in all but one plant
histone gene promoters known to date (Table 1). It is also
present as a module of three kinds of composite elements
(type I, II, and III elements), and interestingly, most
histone genes have one of these three elements (Table 1).
Plant histone genes with the type I or the type II element
are designated type I or type II genes, respectively (Mikami
and Iwabuchi 1993). Here, we propose calling histone
genes with the type HI element as type III genes. That plant
histone genes can be classified based on the elements in the
promoters suggests a mechanism by which the respective
subsets of histone genes are coordinately regulated. Namely, depending on which type(s) of the three Oct-containing
elements is (are) utilized, different subsets of histone proteins could be produced. Since all the H4 genes except
TH091, encoded by both type I and type II genes (Table 1),
have the same amino acid sequence, it is unlikely that each
type of histone genes corresponds to a special function of
their proteins. Rather, the overall amount of histone proteins might be controlled, by modulating the expression
level of a subset of histone genes among the multigene family. The availability and activity of transcription factors interacting with the Hex, TCA, or CCAAT motifs would be
crucial for such regulation and their changes could be relat-
Promoter analysis of a wheat HI gene
ed with tissue-specific and developmental stage-specific expression. Although involvement of the type I element in S
phase-specific transcriptional regulation and meristematic
tissue-specific expression has been shown (Ohtsubo et al.
1997, Terada et al. 1995), little is known about the functions of the type II and III elements in temporal and spatial
regulation of histone genes. Detailed analyses of these
elements will lead to a better understanding of both general
and specific regulation of plant histone genes.
We thank T. Nakayama for his helpful discussion and comments. This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science,
Sports and Culture, and from the Ministry of Agriculture,
Forestry and Fisheries of Japan.
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(Received October 22, 1997; Accepted December 24, 1997)