Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast.

In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.

Mature Myotubes Generated From Human-Induced Pluripotent Stem Cells Without Forced Gene Expression.

Human-induced pluripotent stem cells (hiPSCs) are a promising tool for disease modeling and drug screening. To apply them to skeletal muscle disorders, it is necessary to establish mature myotubes because the onset of many skeletal muscle disorders is after birth. However, to make mature myotubes, the forced expression of specific genes should be avoided, as otherwise dysregulation of the intracellular networks may occur. Here, we achieved this goal by purifying hiPSC-derived muscle stem cells (iMuSC) by Pax7-fluorescence monitoring and antibody sorting. The resulting myotubes displayed spontaneous self-contraction, aligned sarcomeres, and a triad structure. Notably, the phenotype of sodium channels was changed to the mature type in the course of the differentiation, and a characteristic current pattern was observed. Moreover, the protocol resulted in highly efficient differentiation and high homogeneity and is applicable to drug screening.

Discovery of 4,6- and 5,7-Disubstituted Isoquinoline Derivatives as a Novel Class of Protein Kinase C ζ Inhibitors with Fragment-Merging Strategy.

Two chemical series of novel protein kinase C ζ (PKCζ) inhibitors, 4,6-disubstituted and 5,7-disubstituted isoquinolines, were rapidly identified using our fragment merging strategy. This methodology involves biochemical screening of a high concentration of a monosubstituted isoquinoline fragment library, then merging hit isoquinoline fragments into a single compound. Our strategy can be applied to the discovery of other challenging kinase inhibitors without protein-ligand structural information. Furthermore, our optimization effort identified the highly potent and orally available 5,7-isoquinoline 37 from the second chemical series. Compound 37 showed good efficacy in a mouse collagen-induced arthritis model. The in vivo studies suggest that PKCζ inhibition is a novel target for rheumatoid arthritis (RA) and that 5,7-disubstituted isoquinoline 37 has the potential to elucidate the biological consequences of PKCζ inhibition, specifically in terms of therapeutic intervention for RA.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

Telomere-binding proteins play roles in control of replication timing.

Comment on: Tazumi A, et al. Genes Dev 2012; 26:2050-62.

Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast.

DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2'-deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as early-firing origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.