Yuko Imamura

The Yeast Tor Signaling Pathway Is Involved in G2/M Transition via Polo-Kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.

Fredericamycin A Affects Mitochondrial Inheritance and Morphology in Saccharomyces cerevisiae

Fredericamycin A (FMA) is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro, but, its functions in vivo are poorly understood. We identified this compound as an inducer of G1 arrest in the yeast, Saccharomyces cerevisiae. FMA exhibits an IC50 of 24 nM towards the growth of a disruptant of multi-drug resistance genes, W303-MLC30, and its cytotoxicity is a function of the time of exposure as well as drug dose. Addition of 0.8 μM of FMA caused aggregation of mitochondria within 10 min of incubation and the drug induced petites at high frequency after 4 h of incubation. rho − cells were about 20 times more resistant to FMA than isogenic rho + cells. Overexpression of topoisomerase I, a previously suggested target of the drug, did not alleviate the sensitivity of the cells to FMA or the aggregation of mitochondria. Our results suggest that mitochondria are the primary target site of FMA.

The yeast chromatin remodeler Rsc1-RSC complex is required for transcriptional activation of autophagy-related genes and inhibition of the TORC1 pathway in response to nitrogen starvation.

The yeast RSC, an ATP-dependent chromatin-remodeling complex, is essential for mitotic and meiotic growth. There are two distinct isoforms of this complex defined by the presence of either Rsc1 or Rsc2; however, the functional differences between these complexes are unclear. Here we show that the RSC complex containing Rsc1, but not Rsc2, functions in autophagy induction. Rsc1 was required not only for full expression of ATG8 mRNA but also for maintenance of Atg8 protein stability. Interestingly, decreased autophagic activity and Atg8 protein stability in rsc1Δ cells, but not the defect in ATG8 mRNA expression, were partially suppressed by deletion of TOR1. In addition, we found that rsc1Δ impaired the binding between the Rho GTPase Rho1 and the TORC1-specific component Kog1, which is required for down-regulation of TORC1 activity. These results suggest that the Rsc1-containing RSC complex plays dual roles in the proper induction of autophagy: 1) the transcriptional activation of autophagy-related genes independent of the TORC1 pathway and 2) the inactivation of TORC1, possibly through enhancement of Rho1-Kog1 binding.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

RSC Chromatin-Remodeling Complex Is Important for Mitochondrial Function in Saccharomyces cerevisiae.

RSC (Remodel the Structure of Chromatin) is an ATP-dependent chromatin remodeling complex essential for the growth of Saccharomyces cerevisiae. RSC exists as two distinct isoforms that share core subunits including the ATPase subunit Nps1/Sth1 but contain either Rsc1or Rsc2. Using the synthetic genetic array (SGA) of the non-essential null mutation method, we screened for mutations exhibiting synthetic growth defects in combination with the temperature-sensitive mutant, nps1-105, and found connections between mitochondrial function and RSC. rsc mutants, including rsc1Δ, rsc2Δ, and nps1-13, another temperature-sensitive nps1 mutant, exhibited defective respiratory growth; in addition, rsc2Δ and nps1-13 contained aggregated mitochondria. The rsc2Δ phenotypes were relieved by RSC1 overexpression, indicating that the isoforms play a redundant role in respiratory growth. Genome-wide expression analysis in nps1-13 under respiratory conditions suggested that RSC regulates the transcription of some target genes of the HAP complex, a transcriptional activator of respiratory gene expression. Nps1 physically interacted with Hap4, the transcriptional activator moiety of the HAP complex, and overexpression of HAP4 alleviated respiratory defects in nps1-13, suggesting that RSC plays pivotal roles in mitochondrial gene expression and shares a set of target genes with the HAP complex.

Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation

In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.

HIF-2α/ARNT complex regulates hair development via induction of p21Waf1/Cip1 and p27Kip1

The hypoxia‐inducible factors HIF‐1α or HIF‐2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF‐1α/ARNT and HIF‐2α/ARNT complexes activate hypoxia‐inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF‐2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF‐2α/ARNT in hair development using conditional‐knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild‐type mice, HIF‐2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF‐2α and induction of the mammalian cyclin‐dependent kinase inhibitors p21Waf1/Cip1 and p27 Kip1 The results suggest that the HIF‐2α/ARNT complex regulates hair follicle differentiation via induction of p21Waf1/Cip1 and possibly p27Kip1, as p27Kip1 expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.—Imamura, Y., Tomita, S., Imanishi, M., Kihira, Y., Ikeda, Y., Ishizawa, K., Tsuchiya, K., Tamaki, T. HIF‐2α/ARNT complex regulates hair development via induction of p21Waf1/Cip1 and p27Kip1. FASEB J. 28, 2517–2524 (2014). www.fasebj.org

3,6‐Epidioxy‐1,10‐bisaboladiene inhibits G1‐specific transcription through Swi4/Swi6 and Mbp1/Swi6 via the Hog1 stress pathway in yeast

3,6‐Epidioxy‐1,10‐bisaboladiene (EDBD), a bisabolane sesquiterpene endoperoxide compound, was previously isolated from Cacalia delphiniifolia and C. hastata in northern Japan. EDBD has cytotoxic effects and induces apoptosis via phosphorylation of p38 mitogen‐activated protein kinase in human promyelocytic leukemia HL60 cells. However, the mechanism of action of EDBD has not yet been fully elucidated. In this study, we examined the molecular mechanisms of EDBD in the budding yeast Saccharomyces cerevisiae. EDBD arrested the growth of S. cerevisiae cells by suppressing progression from the G1 phase to the S phase and from the G2 phase to the M phase. Moreover, biochemical and genetic analyses revealed that EDBD activated environmental stress–response pathways involving Hog1 and affected Cln3/G1 cyclin activity, thereby inhibiting the expression of SCB‐binding factor and MCB‐binding factor target genes. Our results provided important insights into the functions of EDBD in tumor cells and may facilitate the development of EDBD‐based antitumor therapies.

Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator

Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.