The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying chromatin regulation with high-resolution genome-wide analyses. Since newly generated genome-wide data are often compared with publicly available datasets, expanding our dataset repertoire will be beneficial for the field. Information on transcription start sites (TSSs) determined at base pair resolution is essential for elucidating mechanisms of transcription and related chromatin regulation, yet no datasets that cover two different cell types are available. Here, we present a CAGE (cap analysis of gene expression) dataset for a-cells and α-cells grown in defined and rich media. Cell type-specific genes were differentially expressed as expected, ensuring the reliability of the data. Some of the differentially expressed TSSs were medium-specific or detected due to unrecognized chromosome rearrangement. By comparing the CAGE data with a high-resolution nucleosome map, major TSSs were primarily found in +1 nucleosomes, with a peak approximately 30 bp from the promoter-proximal end of the nucleosome. The dataset is available at DDBJ/GEA.

Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.

The developmental mechanisms of limb buds have been studied in developmental biology as an excellent model of pattern formation. Chick embryos have contributed to the discovery of new principles in developmental biology, as it is easy to observe live embryos and manipulate embryonic tissues. Herein, I outline recent findings and future issues over the next decade regarding three themes, based on my research: limb positioning, proximal–distal limb elongation and digit identity determination. First, how hindlimb position is determined at the molecular level is described, with a focus on the transforming growth factor-β signaling molecule GDF11. Second, I explain how the cell population in the limb bud deforms with developmental progress, shaping the limb bud with elongation along the proximal–distal axis. Finally, I describe the developmental mechanisms that determine digit identity through the interdigits.

The importance of the parent–progeny relationship tracking technique in single-cell analysis has grown with the passage of time. In this study, fundamental image-processing techniques were combined to develop software capable of inferring cell cycle alterations in fission yeast cells, which exhibit equipartition during division. These methods, exclusively relying on bright-field images as input, could track parent–progeny relationships after cell division by assessing the temporal morphological transformation of these cells. In the application of this technique, the software was employed for calculating intracellular fluorescent dots during every stage of the cell cycle, using a yeast strain expressing EGFP-fused Swi6, which binds to chromatin. The results obtained with this software were consistent with those of previous studies. This software facilitated single-cell-level tracking of parent–progeny relationships in cells exhibiting equipartition during division and enabled the monitoring of spatial fluctuations in a cell cycle-dependent protein. This method, expediting the analysis of extensive datasets, may also empower large-scale screening experiments that cannot be conducted manually.

In Saccharomyces cerevisiae, boundaries formed by DNA sequence-dependent or -independent histone modifications stop the spread of the heterochromatin region formed via the Sir complex. However, it is unclear whether the histone modifiers that control DNA sequence-independent boundaries function in a chromosome-specific or -nonspecific manner. In this study, we evaluated the effects of the SAGA complex, a histone acetyltransferase (HAT) complex, and its relationship with other histone-modifying enzymes to clarify the mechanism underlying boundary regulation of the IMD2 gene on the right subtelomere of chromosome VIII. We found that Spt8, a component of the SAGA complex, is important for boundary formation in this region and that the inclusion of Spt8 in the SAGA complex is more important than its interaction with TATA-binding protein and TFIIS. In addition to SAGA, various HAT-related factors, such as NuA4 and Rtt109, also functioned in this region. In particular, the SAGA complex induced weak IMD2 expression throughout the cell, whereas NuA4 induced strong expression. These results indicate that multiple HATs contribute to the regulation of boundary formation and IMD2 expression on the right subtelomere of chromosome VIII and that IMD2 expression is determined by the balance between these factors.

The freezing-thawing (F/T) method for fusing giant unilamellar vesicles (GUVs) can provide substrates, enzymes and membrane material simultaneously and repetitively, and is useful for constructing a recursive model of an artificial cell. However, enzymatic efficiency after F/T is reduced due to rupture of the GUVs and leakage of the inner solution during F/T. Previously, liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a negatively charged lipid, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), showed lower rupture and leakage rates during F/T. In this study, we investigated the effect of POPG on the supply of components required for T7 RNA polymerase reactions via F/T by flow cytometry analysis. We found that the addition of POPG to liposome preparations reduced the efficiency of RNA synthesis. In addition, DNA was concentrated during F/T and RNA synthesis occurred after F/T in liposomes composed of POPC and POPG. Our results provide new insights for more efficient supply of substrates and enzymes by the F/T method, thereby increasing the utility of the F/T method for the construction of recursive bioreactors.