Summary The Cancer Genome Atlas (TCGA) project has analyzed mRNA expression, miRNA expression, promoter methylation, and DNA copy number in 489 high-grade serous ovarian adenocarcinomas (HGS-OvCa) and the DNA sequences of exons from coding genes in 316 of these tumors. These results show that HGS-OvCa is characterized by TP53 mutations in almost all tumors (96%); low prevalence but statistically recurrent somatic mutations in 9 additional genes including NF1, BRCA1, BRCA2, RB1, and CDK12; 113 significant focal DNA copy number aberrations; and promoter methylation events involving 168 genes. Analyses delineated four ovarian cancer transcriptional subtypes, three miRNA subtypes, four promoter methylation subtypes, a transcriptional signature associated with survival duration and shed new light on the impact on survival of tumors with BRCA1/2 and CCNE1 aberrations. Pathway analyses suggested that homologous recombination is defective in about half of tumors, and that Notch and FOXM1 signaling are involved in serous ovarian cancer pathophysiology.

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

PDCD2 is a conserved eukaryotic protein implicated in cell cycle regulation by virtue of its interactions with HCFC1 and the NCOR1/SIN3A corepressor complex. Pdcd2 transcripts are enriched in ES cells and other somatic stem cells, and its ortholog is essential for hematopoietic stem cell maintenance in Drosophila. To characterize the physiological role(s) of mammalian PDCD2, we created a disruption allele in mice. Pdcd2−/− embryos underwent implantation but did not undergo further development. Inner cell masses (ICMs) from Pdcd2−/− blastocysts failed to outgrow in vitro. Furthermore, embryonic stem cells (ESCs) require PDCD2 as demonstrated by the inability to generate Pdcd2−/− ESCs in the absence of an ectopic transgene. Upon differentiation of ESCs by retinoic acid treatment or LIF deprivation, PDCD2 levels declined. In conjunction with prior studies, these results indicate that in vivo, PDCD2 is critical for blastomere and ESC maintenance by contributing to the regulation of genes in a manner essential to the undifferentiated state of these cells.

BackgroundForward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.ResultsWe report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.ConclusionThis collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.

Summary Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation [1–3]. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to one megabase from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing shows that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment, and the role of programmed changes in chromosome structure in replication control are discussed.