In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond. Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown. Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE. Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8p G116 , Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction. Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition). It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p. In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy.

Although the role of p21 Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21 Waf1/Cip1 gene expression in vivo, we attempted to visualize the dynamics of p21 Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21 Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21 Waf1/Cip1 gene expression in vivo. By monitoring and quantifying the p21 Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21 Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21 Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21 Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21 Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21 Waf1/Cip1 knockout mice, has uncovered a novel role for the p21 Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.aging ͉ cell cycle ͉ hair cycle ͉ imaging

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.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

The ligand-receptor-mediated intercellular communication system plays important roles in coordinating developmental and physiological events in multicellular organisms. In plants, CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides and their cognate receptors are thought to be involved in various aspects of the plant life cycle. Although the importance of this communication is broadly recognized, most CLE peptides are yet to be functionally characterized. A major problem in research on small signaling peptide-encoding genes is the limited number of loss-of-function mutants available due to their small gene size. CRISPR/Cas9-mediated gene targeting has the potential to overcome this problem, as it can be used to generate targeted mutations in essentially any gene, regardless of size. Here we generated a series of mutants of CLE-peptide-encoding genes. Newly generated clv3 and cle40 mutants reproduced the expected mutant phenotypes in the shoot apical meristem and root meristem, respectively. Our results show that CRISPR/Cas9-mediated gene targeting is a powerful tool for genetic analyses, even of small genes. We also report a novel mutant for CLE44 [which is thought to encode a tracheary elements differentiation inhibitory factor (TDIF)] and show that CLE44 contributes to vascular development. The bioresources presented here will be a powerful tool for further characterization of CLE peptides.

Cells may exchange information with other cells and tissues by exerting forces on the extracellular matrix (ECM). Fibronectin (FN) is an important ECM component that forms fibrils through cell contacts and creates directionally biased geometry. Here, we demonstrate that FN is deposited as pillars between widely separated germ layers, namely the somitic mesoderm and the endoderm, in quail embryos. Alongside the FN pillars, long filopodia protrude from the basal surfaces of somite epithelial cells. Loss-of-function of Ena/VASP, α5β1-integrins or talin in the somitic cells abolished the FN pillars, indicating that FN pillar formation is dependent on the basal filopodia through these molecules. The basal filopodia and FN pillars are also necessary for proper somite morphogenesis. We identified a new mechanism contributing to FN pillar formation by focusing on cyclic expansion of adjacent dorsal aorta. Maintenance of the directional alignment of the FN pillars depends on pulsatile blood flow through the dorsal aortae. These results suggest that the FN pillars are specifically established through filopodia-mediated and pulsating force-related mechanisms.

Molecular epidemiological investigation was conducted among injecting drug users (IDUs) (n = 11) and heterosexuals (n = 15) in Kunming, Yunnan Province of China. HIV-1 genotypes were determined based on the nucleotide sequences of 2.6-kb gag-RT region. The distribution of genotypes among IDUs was as follows: CRF07_BC (5/11) and CRF08_BC (5/11); subtype B' (1/11). Similarly, a majority of Kunming heterosexuals (14/15) were infected with CRF07_BC (4/15), CRF08_BC (6 /15), or subtype B' (4/15), known to predominate among IDUs in China. This contrasts with trends in the coastal regions of China and surrounding southeastern Asian countries, where CRF01_AE predominates among heterosexuals. The heterosexual HIV-1 epidemic in Kunming thus appears to derive from the local IDU epidemic. Of note, subtype B' was the most prevalent strain among heterosexuals before 1997, while CRF07_BC and CRF08_BC became predominant in 2002, indicating a transition of HIV-1 genotype distribution between the early and the more recent samples from Kunming heterosexuals.

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.