Establishment of dsDNA-dsDNA interactions by the condensin complex

Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a “diffusion capture” model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase”) was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome

Efficacy and safety of monotherapy with the novel sodium/glucose cotransporter-2 inhibitor tofogliflozin in Japanese patients with type 2 diabetes mellitus : A combined Phase 2 and 3 randomized, placebo-controlled, double-blind, parallel-group comparative study

BACKGROUND: In recent years, several oral antidiabetic drugs with new mechanisms of action have become available, expanding the number of treatment options. Sodium/glucose cotransporter-2 (SGLT2) inhibitors are a new class of oral antidiabetic drugs with an insulin-independent mechanism promoting urinary glucose excretion. We report the results of a combined Phase 2 and 3 clinical study (Japic CTI-101349) of the SGLT2 inhibitor tofogliflozin (CSG452, RG7201) in Japanese patients with type 2 diabetes mellitus. METHODS: The efficacy and safety of tofogliflozin were assessed in this multicenter, placebo-controlled, randomized, double-blind parallel-group study involving 230 patients with type 2 diabetes mellitus with inadequate glycemic control on diet/exercise therapy. Between 30 October 2010 and 28 February 2012, patients at 33 centers were randomized to either placebo (n = 56) or tofogliflozin (10, 20, or 40 mg; n = 58 each) orally, once daily for 24 weeks. The primary efficacy endpoint was the change from baseline in HbA(1c) at week 24. RESULTS: Overall, 229 patients were included in the full analysis set (placebo: n = 56; tofogliflozin 10 mg: n = 57; tofogliflozin 20 and 40 mg: n = 58 each). The least squares (LS) mean change (95% confidence interval) from baseline in HbA(1c) at week 24 was −0.028% (−0.192 to 0.137) in the placebo group, compared with −0.797% (−0.960 to −0.634) in the tofogliflozin 10 mg group, −1.017% (−1.178 to −0.856) in the tofogliflozin 20 mg group, and −0.870% (−1.031 to −0.709) in the tofogliflozin 40 mg group (p < 0.0001 for the LS mean differences in all tofogliflozin groups vs placebo). There were also prominent decreases in fasting blood glucose, 2-h postprandial glucose, and body weight in all tofogliflozin groups compared with the placebo group. The main adverse events were hyperketonemia, ketonuria, and pollakiuria. The incidence of hypoglycemia was low. Furthermore, most adverse events were classified as mild or moderate in severity. CONCLUSIONS: Tofogliflozin 10, 20, or 40 mg administered once daily as monotherapy significantly decreased HbA(1c) and body weight, and was generally well tolerated in Japanese patients with type 2 diabetes mellitus. Phase 3 studies were recently completed and support the findings of this combined Phase 2 and 3 study. TRIAL REGISTRATION: This study was registered in the JAPIC clinical trials registry (ID: Japic CTI-101349)

Swi1^Timeless Prevents Repeat Instability at Fission Yeast Telomeres

<div><p>Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1^Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in <i>swi1</i>Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1^Timeless in regulation of telomere stability in cancer cells.</p></div

Swi1 loss causes DNA damage at telomeres.

<p><b>(A)</b> Genome-wide (ChIP-seq) analysis of Rad52 distribution at the subtelomeres (left arm of chromosome 1) in asynchronous cultures of wild-type (green) and <i>swi1Δ</i> (blue) cells. Enrichment of Rad52 is displayed as enrichment scores (y-axis). Chromosome coordinates [x-axis, in megabases, (Mb)] were downloaded from the <i>S</i>. <i>pombe</i> Genome Project (Sanger Center: <a href="http://www.sanger.ac.uk/Projects/S_pombe" target="_blank">www.sanger.ac.uk/Projects/S_pombe</a>). The strains used for our experiments are both heterothallic <i>h</i>^<i>+</i> strains. <b>(B)</b> Box plots for log2 ratios of Rad52 enrichment at subtelomeric regions vs other regions. The boxes were limited by the 25th and 75th percentiles, with the black lines representing the median ratios between <i>swi1</i>Δ and wild-type. The whiskers are extended out to the most extreme data points that are at most 1.5 times the interquartile range from the box. The black circles represent outliers. The <i>p</i>-value was evaluated by Mann-Whitney U test. <b>(C)</b> ChIP assays showing Rad52 enrichment at telomeres in wild-type and <i>swi1</i>Δ cells. Rad52-12Pk was immunoprecipitated from the indicated cells, and associated DNA was subjected to competitive multiplex PCR to amplify DNA fragments from the <i>TAS1</i> region and a gene-free region (GFR2), which was used as an internal amplification control. Chromatin association of Rad52-12Pk at <i>TAS1</i> was presented as relative enrichment over the association at GFR2. Data are expressed as the mean of three independent experiments. Error bars represent the standard deviation. <i>p</i>-value was determined by two-tailed Student's t-test. <b>(D-E-F)</b> Wild-type and <i>swi1Δ</i> cells expressing Rad52-YFP and Taz1-mCherry were grown in minimal medium at 25°C until mid-log phase. Nuclei (n = 102) were analyzed for the percentage of TIF-positive nuclei in (D) as well as for the total number of Rad52-YFP and Taz1-mCherry foci in (E). Accumulation of Rad52-YFP foci (E) and a significant increase in TIF-positive nuclei were observed in the <i>swi1Δ</i> cells (D). Data shown is the mean of three independent experiments. Error bars represent the standard deviation. <i>p</i>-values were determined by two-tailed Student's t-test. (F) Representative microscopic images. Pink arrows indicate TIFs.</p