HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance

Influence of locational states of submicron fibers added into matrix on mechanical properties of plain-woven Carbon Fiber Composite

The aim of this study was to show the influence of locational states of submicron fibers added into epoxy matrix on mechanical properties of modified plane-woven carbon fiber reinforced plastic (CFRP). To change the locational states of submicron fibers, two kinds of fabrication processes were applied in preparing specimen by hand lay-up method. Submicron fibers were simply added into epoxy resin with ethanol after they were stirred by a dispersion process using homogenizer to be located far from the interface between reinforcement and matrix. In contrast, submicron fibers were attached onto the carbon fibers by injecting from a spray nozzle accompanying with ethanol to be located near the interface, after they were tentatively contained in ethanol. The plain-woven CFRP plates were fabricated by hand lay-up method and cured at 80 degree-C for 1 hour and then at 150 degree-C for 3 hours. After curing, the plain-woven CFRP plates were cut into the dimension of specimen. Tensile shear strength and Mode-II fracture toughness of CFRP were determined by tensile lap-shear test and End-notched flexure(ENF) test, respectively. When submicron fibers were located far from the interface between carbon fibers and epoxy resin, tensile shear strength and Mode-II fracture toughness of CFRP were improved 30% and 18% compared with those of unmodified case. The improvement ratio in modified case was rather low (about few percentages) in the case where submicron fibers were located near the interface. The result suggested that crack propagation should be prevented when submicron fibers were existed far from the interface due to the effective stress state around the crack tip

Sex Specification and Heterogeneity of Primordial Germ Cells in Mice.

In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells

Biological significance of female and male-specific PGC-expressed genes.

<p><b>(A)</b> GO enrichment analysis of FSGs and MSGs. The most highly enriched biological processes based on their respective gene counts are shown (Fisher’s exact test: cut-off < 0.1). <b>(B)</b> Pathway analysis of all transcript lists in female and male PGCs. The lists indicate enriched pathways observed among female and male transcripts, as determined using DAVID (Fisher’s exact test: cut-off <0.1). Sex-specific pathways are highlighted in pink and blue, which denote female- and male-specific, respectively.</p

DNA methylation and histone modifications in female- and male-specific PGC-expressed genes.

<p><b>(A)</b> Average methylation levels of TSS and the shore values (±5 kb of the TSS) in female- and male-specific PGC-expressed genes. Our previous methylation data of PGCs were obtained from the DDBJ database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144836#pone.0144836.ref012" target="_blank">12</a>]. <b>(B)</b> Occupancy of H3K4me3- and H3K27me3-bound TSS and TTS shore regions (±5 kb of the TSS and TTS) in female- and male-specific PGC-expressed genes. <b>(C)</b> Proportion of genes showing histone-modification patterns. The number of genes classified into each histone-modification pattern is shown. <b>(D)</b> Three typical histone-modification patterns. The red regions indicate ChIP/input enrichment-peak regions that were calculated using the DROMPA peak-calling program.</p

Single-cell transcriptome analysis of E13.5 female and male PGCs.

<p><b>(A)</b> Bi-dimensional <b>PCA</b> of E13.5 female and male single-PGC gene expression patterns. The first principal component (PC1) captures 4.93% of the gene expression variability and the second principal component (PC2) captures 4.25%. The red and blue spheres represent single female (n = 67) and male (n = 77) PGCs, respectively. <b>(B)</b> Hierarchical clustering analysis of transcriptome data sets (FSG: 651, MSG: 428) of female and male single PGCs. <b>(C)</b> Heat map of the relative expression of apoptotic process-related genes (n = 15). <b>(D)</b> Heat map of the relative expression of meiotic and mitotic cell cycle-related genes (n = 26). <b>(E)</b> Relation between apoptosis and meiotic and mitotic cell cycle in female PGCs. The numbers indicate the number of PGCs.</p

Identification of female and male PGC-specific-expressed genes.

<p><b>(A)</b> Top: scatter plot of female and male PGC samples. The green lines indicate a 2-FC in the average expression levels between the 2 samples. Bottom: volcano plot of differentially expressed genes in female and male PGC samples. Red spheres indicate statistically significant genes. FSGs (651) and MSGs (428). <b>(B)</b> Top 20 differentially expressed genes in female- and male-specific PGCs. Expression levels are shown in log2 values. The genes are ranked in the order of decreasing log FC values. <b>(C)</b> Pie chart showing the composition and quantity of each reference gene type in the UCSC Genome Browser and Ingenuity Pathway analysis tool for FSGs and MSGs.</p