(1)from University of Toronto)
(2)from Kyoto University)
Genes encoding four types of core histones (H2A, H2B, H3, H4) and a linker histone (H1) are coordinately expressed during S phase of the cell cycle. To understand how this coordinate expression is regulated, we have been continuously studying the transcriptional regulation of wheat histone genes.
Within the promoter region of an H3 gene (TH012), three types of positive cis-acting elements, the hexamer (ACGTCA), the octamer (CGCGGATC) and the nonamer (CATCCAACG) have been found. The hexamer and the octamer constitute a sequence unit designated as type I element. This combined sequence is often found within the promoter region of plant histone H2B, H3 and H4 genes.
We have also found several sequence-specific DNA-binding proteins interacting with the type I element; HBP-1a and HBP-1b specific for the hexamer, and OBRFs for the octamer. Analyses of cloned cDNAs for HBP-1a and HBP-1b revealed that they have the characteristic feature of bZIP (basic region/leucine zipper) type transcription factors.
To analyze the temporal patterns of plant histone gene expression during the cell cycle, we have established the cell cycle synchronization system using suspension culture of rice Oc cells and the inhibitor of DNA synthesis, aphidicolin. S1 analyses of mRNAs transcribed from
chimeric genes composed of the H3 promoter and protein coding region of B-glucuronidase gene revealed that the type I element is necessary and sufficient for the S phase-specific activation of the H3 gene expression.
I. Auxin-inducible expression of the wheat histone H3 gene
To define the functions of the type I element in vivo, we introduced the H3/GUS chimeric gene (-185WTH3/GUS) into Arabidopsis plants via Agrobacterium Ti plasmid, then analyzed their expressions in roots of regenerated plants. Histochemical staining experiment showed that the WTH3/GUS gene is expressed at root apical meristem and vascular bundle (Fig. 1A) whereas expression in the latter region was not observed when we examined using regenerated roots of transgenic rice plants. Expression in vascular bundle may occur in the dividing cells that constitute secondary vascular bundle specific for dicots and gymnosperms. We concluded that the H3 gene expression in roots is detected only in dividing cells whose cell cycle is actively progressing.
To determine whether the type I element is also involved in the ineristem-specific gene expression, transgenic plants harboring mutant genes, HexM, OctM, and HexM/OctM were tested. Base-substitutions introduced into the hexamer or the octamer abolished the expression in roots (data not shown), suggesting that the type I element is necessary for the H3 gene expression in dividing cells.
Since auxin acts as a mitogen, progression of the cell cycle and expression of cell cycle-dependent genes such as histone genes would be expected to be regulated by auxin. To address this possibility, we examined the effect of exogenously provided auxin on the meristem-specific expression of the H3/GUS genes. As shown in Fig.1B, expression of the WTH3/GUS gene was enhanced in both root apical meristem and vascular bundle. This result indicates that the action of auxin is involved in the signal transduction pathway from the passage of G1/S boundary to the H3 gene expression. Detailed analyses of the H3 promoter sequence that functions in this induction pathway are currently under way.
II. Functional analysis of HBP-1b(c38)
When the HBP-1b(c38) expression plasmid was co-transfected into tobacco mesophyll protoplasts with reporter plasmids containing the binding site of HBP-1b(c38), expression of the reporter gene was repressed. However, when the hexamer (a binding site of HBP-1b) was mutated, HBP-1b(c38) acted as an activator. This result represents a dual function of HBP-1b(c38) through direct binding to DNA and protein-protein interactions with the octamer-binding protein(s) or some other transcription fac-tors. In addition, we investigated the functional difference between HBP-1b(c38) and its tobacco homologue, TGA-1a, in this expression system. Swapping experiments aiming at the functional regions of HBP-1b(c38) and TGA- 1a showed that the activation of gene expression through the hexamer motif depends on the acidic region of TGA-1a.
To elucidate the functional roles of HBP-1b(c38) in plant development, we made transgenic Arabidopsis harboring the plasmids that overexpress HBP-1b(c38) or its antisense RNA and observed morphological effects of transgenic plants. Transformants overexpressing HBP-1b(c38) showed significant morphological changes such as bent stems, leafless plants, and abnormal production of flowers. This result suggests that HBP-1b(c38) is involved in the regulation of cell fate determination, flower development and/or other growth control mechanisms.
III. S phase-specific expression of the wheat histone H1 gene
The genomic clones, TH315 and TH325, for wheat H1 genes, have not the type I element within their promoter region. Instead, they have another type of characteristic sequence unit composed of the octamer and the CAAT-box. This sequence, designated as the type III element, is found not only within the promoter region of wheat histone H1 genes but also within those of Arabidopsis (AtH1-1) and tomato H1 genes and wheat H2B genes (TH123 and TH153). Conservation of the order and the spacing (14 bp) between the two components (the octamer and CAAT) represents the possibility that the type III element is involved in the S phase-specific regulation of H1 and H2B gene expression.
Transient expression analyses of 5'-deletion and base-substitution mutants of H1 promoter/GUS chimeric genes revealed that the type III element acts as a positive cis-acting element of the H1 (TH315) gene.
To determine whether the type III element is involved in the regulation of S phase-specific gene expression, and to understand how the regulation of core histone gene expression during the S phase is different from that of H1 genes, we analyzed the expression of the H1/GUS chimeric genes in transformed rice cells. S1 analyses of the H1/GUS mRNA from synchronized culture of transformed rice cells showed that the peak of H1/GUS mRNA accumulation appeared slightly later (by an hour) than that of H3/GUS mRNA. In addtion, when the type III element of this promoter was deleted by truncation from its 5' end, the S phase-specific change of mRNA level was no longer observed. These results suggest the difference in the regulatory mechanism between the H1 gene and H3 gene, and also represent the contribution of the type III element to the S phase-specific regulation of the H1 gene expression.
Publication List:
Mikami, K., Sakamoto, A. and lwabuchi, M. (1994) The HBP-1 family of wheat basic/leucine zipper proteins interacts with overlapping cis-acting hexamer motifs of plant histone genes. J. Biol. Chem.269, 9974-9985.
Morio, T., Takeuchi, I. and Tasaka, M. ( 1994) Cooperation of positively and negatively acting promoter elements determines prespore-specific transcription of Dp87 gene in Dictyostelium. Mech. Dev.45, 59-72.
Nakao, H., Yamamoto, A., Takeuchi, I. and Tasaka, M. (1994) Dictyostelium prespore-specific gene Dp87 encodes a sorus matrix protein. J. Cell Sci.107, 397-403.
Ohtsubo, N. and lwabuchi, M. (1994) The conserved 3'-flanking sequence, AATGGAAATG, of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA, Nucl. Acids Res.22, 1052-1058.
Saran, S., Nakao, H., Tasaka, M., Iida,
H., Tsuji, F.I., Nanjundiah, V. and Takeuchi, I. (1994) Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyiish apoaequorin cDNA. FEBS Lett.337, 43-47.
National Insitute for Basic Biology
