A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TMAgtsl) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TMAgtsl and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TMAgtsl and TMpGTS1 were about 20%o shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TMAgtsl cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.Among the genes known to affect biological clocks, the period gene (per) of Drosophila melanogaster (24, 37) and the frequency gene (frq) of Neurospora crassa (31) were the first to be isolated and sequenced. Characteristically, the amino acid sequences deduced from the nucleotide sequences of both genes each contained a Gly-Thr/Ser repeat which is shared by proteoglycans comprising a polysaccharide attachment site (31,37,43). Although the GT repeats are polymorphic in length and in sequence among the species of Drosophila (10,11,16,34,35,48,51), the presence of the GT repeat in the clockaffecting genes from evolutionarily remote species, Drosophila and Neurospora species, implies that this sequence plays an important role in most, if not all, clock-affecting proteins.The nature of per mutant phenotypes suggested that the gene product is an integral component of the circadian clock: mutations either shorten (pers), lengthen (per'), or abolish (per0) not only the period of circadian rhythms (19) but also the rhythm with a much shorter period during the male courtship song (26,52). Furthermore, the period of the circadian rhythm of D. melanogaster changes depending upon the gene dose (4,12,45

We previously reported that the GTS1 product, Gts1p, plays an important role in the regulation of heat tolerance of yeast under glucose-limited conditions in either batch or continuous culture. Here we show that heat tolerance was decreased in GTS1-deleted and increased in GTS1-overexpressing cells under glucose-derepressed conditions during the batch culture and that the disruption of SNF1, a transcriptional activator of glucose-repressible genes, diminished this effect of GTS1. Intracellular levels of Hsp104 and trehalose, which were reportedly required for the acquisition of heat tolerance in the stationary phase of cell growth, were affected in both GTS1 mutants roughly in proportion to the gene dosage of GTS1, whereas those of other Hsps were less affected. The mRNA levels of genes for Hsp104 and trehalose-6-phosphate synthase 1 changed as a function of GTS1 gene dosage. The Q-rich domain of Gts1p fused with the DNA-binding domain of LexA activated the transcription of the reporter gene LacZ, and Gts1p lacking the Q-rich domain lost the activation activity of HSP104 and TPS1. Furthermore, Gts1p bound to subunits of Snf1 kinase, whereas it did not bind to DNA. Therefore, we suggested that GTS1 increases heat tolerance by mainly activating Snf1 kinase-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth.We reported that the GTS1 gene shows pleiotropic effects on yeast in batch cultures, including the effect on heat tolerance as a function of gene dosage (1); overexpression of GTS1 increases and deletion of GTS1 decreases the heat tolerance of yeast in the stationary phase of growth, whereas no such effects were found in exponentially growing cells. Independently, Bossier et al. (2) isolated GTS1 from cDNA library-transformed yeast which re-grow after lethal heat shock and found that overexpression of GTS1 results in an unchanged growth rate at 37°C compared with 28°C. On the other hand, we reported that GTS1 is involved in regulating ultradian oscillations of energy metabolism in continuous cultures under aerobic and glucose-limited conditions and in the coupling of oscillations of cellular responses to various stress conditions, including heat with the energy metabolism oscillation (3-5). These results suggested that the gene product Gts1p plays an important role in the regulation of heat and other stress responses under glucose-limited or -depleted conditions in either batch or continuous culture.

We reported previously that Gts1p regulates oscillations of heat resistance in concert with those of energy metabolism in continuous cultures of the yeast Saccharomyces cerevisiae by inducing fluctuations in the levels of trehalose, but not in those of Hsp104 (heat shock protein 104). Further, the expression of TPS1, encoding trehalose-6-phosphate synthase 1, and HSP104 was activated by Gts1p in combination with Snf1 kinase, a transcriptional activator of glucose-repressible genes, in batch cultures under derepressed conditions. Here we show that, in continuous cultures, the mRNA level of TPS1 increased 6-fold in the early respiro-fermentative phase, while that of HSP104 did not change. The expression of SUC2, a representative glucose-repressible gene encoding invertase, also fluctuated, suggesting the involvement of the Snf1 kinase in the periodic activation of these genes. However, this possibility was proven to be unlikely, since the oscillations in both TPS1 and SUC2 mRNA expression were reduced by approx. 3-fold during the transient oscillation in gts1Delta (GTS1-deleted) cells, in which the energy state determined by extracellular glucose and intracellular adenine nucleotide levels was comparable with that in wild-type cells. Furthermore, neither the mRNA level nor the phosphorylation status of Snf1p changed significantly during the oscillation. Thus we suggest that Gts1p plays a major role in the oscillatory expression of TPS1 and SUC2 in continuous cultures of Saccharomyces cerevisiae, and hypothesized that Gts1p stabilizes oscillations in energy metabolism by activating trehalose synthesis to facilitate glycolysis at the shift from the respiratory to the respiro-fermentative phase.

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Smad proteins are transcriptional regulators activated by TGF-␤. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5-GTCTAGAC-3) and CAGA motifs (5-AGCCAGACA-3 or 5-TGTCTGGCT-3). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-␤. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 16H05150 (to K. Miyazawa) and 18K06626 (to Y. I.), JSPS Core-to-Core Program "Cooperative International Framework in TGF-␤ Family Signaling," the Fugaku Trust for Medicinal Research (to K. Miyazawa), the Mitsubishi Science Foundation (to K. Miyazawa), and the Terumo Foundation for Life Science and Arts (to K. Miyazawa). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1-S7 and Figs. S1-S6. Raw data of CASTing analysis have been deposited in the DDBJ BioProject database under BioProject accession number DRA007794. The ChIP-seq data reported in this paper have been submitted to the Gene Expression Omnibus (GEO) database under GEO accession number GSE121254.