NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)
- Masaki Iwabuchi
- Associate Professor:
- Masao Tasaka
- Research Associates:
- Koji Mikami
- JSPS Postdoctoral Fellow:
- Hisabumi Takase
- Visiting Scientist:
- Shweta Saran (from University of Delhi, India)
- Graduate Students:
- Takuya Ito (from Kyoto Univercity)
Ken-ichiro Taoka (from Kyoto Univercity)
Cell division is the most fundamental event in cell growth and cell
differentiation during development. One of powerfull approaches for
understanding of the molecular mechanisms of cell division is to investigate
the gene expression regulated in a cell cycle-dependent manner. Representative
examples of eukaryotic cell cycle-dependent genes are histone genes, expressed
mainly in the S phase during the cell cycle. We have identified the cis-acting
hexamer (ACGTCA) motif involved in S phase-specific transcription of the wheat
histone H3 (TH012) gene, and bZIP-class trans-acting factors specific to the
hexamer motif. Of bZIP proteins, HBP-Ia(17) and HBP-1b(c38) are known to be
essential for controlling periodic transcription of the H3 gene, because genes
for two proteins are also regulated in a cell cycle-dependent manner. It is,
therefore, likely that HBP-1a(17) and HBP-1b(c38) function as regulators of
cell division in plants. In 1993, we focused our attention on the functional
significance of bZIP proteins during plant development (summarized in the
sections I and II).
We also have been continuously studying the transcriptional regulation of
wheat histone genes from a different viewpoint. Based on the function in
chromatin formation, histones are classified into core (H2A, H2B, H3, and H4)
and linker (H1) proteins. Despite differences in their functions, core and
linker histone genes are coordinately expressed in S phase during the cell
cycle. To understand how a coordinate expression of core and linker histone
genes is regulated, we investigated the regulatory mechanisms of the wheat
histone H1 (TH315) gene (surmmarized in the section III).
I. Regulation of a gene encoding transcription factor HBP-Ia(17) in
- To analyze how the HBP-1a(17) and HBP-1b(c38) genes are regulated in
vivo, we have produced transgenic Arabidopsis plants carring chimeric
genes which are composed of the HBP-1a(17) or HBP-1b(c38) promoter and
the B-glucronidase (GUS) coding sequence. The expression patterns of
the chimeric genes were histochemocally analyzed during development
from germination to seed production. Unfortunately, the HBP-1b(c38)
promoter/GUS chimeric gene was not expressed in any transgenic plants.
We, therefore, examined the spacial and temporal expression pattern of
the HBP-1a(17) promoter/GUS fusion gene.
- In germinating seedlings, the HBP-1a(17) promoter/GUS fusion gene was
expressed in only cotyledons and thereafter in cotyledons and first
leaves (Figs. 1A and 1B). At eight days after germination, GUS activity
was detected mainly in first and second leaves, although weak GUS
activity was still observed in expanded cotyledons (Figs. 1C). The gene
expression seemed to decrease as the leaves became older. As plants
progressed from vegitative to productive growth, GUS activity was not
detected in floral organs, stems, cauline leaves and axillary buds
(Figs. 1D and 1E), indicating that the HBP-1a(17) promoter is activated
in only developing young leaves in the vegitative growth stage.
- We next investigated the effect of light on the HBP-1a(17) expression
during germination. When seven-day-old seedlings grown in light were
placed in the dark for a week, GUS activity in young leaves was
decreased (Fig. 1F). Since dark-grown seedlings showed no GUS activity
in any organs (data not shown), the above result indicated that the
expression of the HBP-1a(17) gene is controlled by light. This
conclusion is supported by the fact that the HBP-1a(17) promoter has
several characteristic sequences such as boxII, I-box, G-box and GATA
motif, all of which are known to be involved in the light-regulated
transcription. Since HBP-1a(17) can specifically interact with the G-box
, we supposed that HBP-1a(17) is implicated in the transcriptional
regulation of lightinducible genes in the early stage of
photomorphogenesis as well as cell cycle-dependent gene expression. This
possibility is beeing checked using plants produced by mating
HBP-1a(17)-expressing transgenic Arabidopsis with several
phytochrome-deficent Arabidopsis mutants.
II. Functional analysis of HBP-1b(c38)
- When the HBP-1b(c38)-expression plasmid was co-transfected into tobacco
BY-2 cells with reporter plasmids containing the binding sites of
HBP-1b(c38), expression of the reporter gene was repressed. This
suggests that HBP-1b(c38) may act as a transcriptional repressor. To
elucidate the functional roles of HBP-1b(c38) in plant development, we
have been trying to produce transgenic Arabidopsis which can
overexpress Arabidopsis HBP-1b(c38)-homologue or its antisense RNA.
III. Cis-control elements for S phase-specific expression of the
wheat histone H1 gene
- To study cell cycle-dependent transcription of the wheat H1 gene,
cultured rice cells were transformed with a chimeric gene which
consists of the GUS coding sequence and the 5' upstream sequence of the
H1 gene spanning from -771 to +74. When cell cycle progression of the
stable transformants was partially synchronized by aphidicolin
treatment, the GUS mRNA level was peaked at 2hr after the removal of
drug and then gradualy decreased (Fig. 2A). Considering a result of DNA
synthesis rate in aphidicolin-treated cells, we concluded that the
wheat H1 gene expression is regulated in an S phase-specific manner in
- The H1 promoter that was 5'-deleted to the position -153 still had the
ability to regulate S phase-specific transcription, although the level
of the GUS mRNA transcribed from the 5'-deleted promoter appeared to be
maximum at 1hr after the removal of aphidicolin (Fig. 2B). The proximal
promoter region (-153 to +74) contains several characteristic sequences,
conserved in the Arabidopsis H1 promoters as well. We are now doing more
detailed experiments to identify the cis-element directing S
phase-specific transcription of the wheat H1 gene.
(1) Original articles
- Minami, M., Huh, G. -H., Yang, P. and Iwabuchi, M. (1993) Coordinate
gene expression of five subclass histones and the putative
transcription factors, HBP-1a and HBP-1b, of histone genes in wheat.
Plant Mol. Biol. 23, 429-434.
- Nasuda, S., Liu, Y.-G., Sakamoto, A., Nakayama, T., Iwabuchi, M. and
Tsunewaki, K. (1993) Chromosomal 10cations of the genes for histones
and a histone gene-binding protein family HBP-1 in common wheat. Plant
Mol. Biol. 22, 603-614.
- Ohtsubo, N., Nakayama, T., Terada, R., Shimamoto, K. and Iwabuchi, M
(1993) Proximal promoter region of the wheat histone H3 gene confers S
phase-specific gene expression in transformed rice cells. Plant Mol.
Biol. 23, 553-565.
- Ozaki, T., Nakao, H., Takeuchi, I. and Tasaka, M. (1993) Developmental
regulation of transcription of a novel prespore-specific gene (Dp87) in
Dictyosterium discoideum. Development 117, 1299-1308.
- Sakamoto, A., Minami, M., Huh, G. -H. and Iwabuchi, M. (1993) The
putative zing-finger protein WZF1 interacts with a cis-acting element
of wheat histone genes. Eur. J. Biochem. 217, 1049-1056.
- Terada, R., Nakayama, T., Iwabuchi, M. and Shimamoto, K. (1993) A wheat
histone H3 promoter confers cell division-dependent and -independent
expression of the gus A gene in transgenic rice plants. PlantJ. 3,
(2) Reviews etc.
- Mikami, K. and Iwabuchi, M. (1993) Regulation of cell cycle-dependent
gene expression. In Control of Plant Gene Expression (Verma, D.P.S.
ed.), CRC Press, Boca Raton, FL, pp.51-68.
- Mikami, K., Takase, H. and Iwabuchi, M. (1993) Gel mobility shift
assay. In Plant Molecular Biology Manual (G. Jonker, ed.), Kluwer Acad.
Publ., in press.
- Nakayama, T. and Iwabuchi, M. (1993) Regulation of wheat histone gene
expression. Critical Reviews in Plant Science 12, 97-110.
- Takase, H. and Iwabuchi, M. (1993) Transcriptional and
post-transcriptional regulation of the expression of wheat histone
genes; Cis-acting elements and trans-acting factors. J. Plant Res.
Special Issue 3, 37-50