Masaki Iwabuchi

Associate Professor:
Masao Tasaka

Research Associates:
Koji Mikami
Takefumi Kawata

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 transgenic Arabidopsis

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 rice cells.

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.

Publication List:

(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, 241-252.

(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