Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism

Gene amplification plays important roles in the progression of cancer and contributes to acquired drug resistance during treatment. Amplification can initiate via dicentric palindromic chromosome production and subsequent breakage–fusion–bridge cycles. Here we show that, in fission yeast, acentric and dicentric palindromic chromosomes form by homologous recombination protein-dependent fusion of nearby inverted repeats, and that these fusions occur frequently when replication forks arrest within the inverted repeats. Genetic and molecular analyses suggest that these acentric and dicentric palindromic chromosomes arise not by previously described mechanisms, but by a replication template exchange mechanism that does not involve a DNA double-strand break. We thus propose an alternative mechanism for the generation of palindromic chromosomes dependent on replication fork arrest at closely spaced inverted repeats.     

Leaping forks at inverted repeats

Genome rearrangements are often associated with genome instability observed in cancer and other pathological disorders. Different types of repeat elements are common in genomes and are prone to instability. S-phase checkpoints, recombination, and telomere maintenance pathways have been implicated in suppressing chromosome rearrangements, but little is known about the molecular mechanisms and the chromosome intermediates generating such genome-wide instability. In the December 15, 2009, issue of Genes & Development, two studies by Paek and colleagues (2861–2875) and Mizuno and colleagues (pp. 2876–2886), demonstrate that nearby inverted repeats in budding and fission yeasts recombine spontaneously and frequently to form dicentric and acentric chromosomes. The recombination mechanism underlying this phenomenon does not appear to require double-strand break formation, and is likely caused by a replication mechanism involving template switching.     

Structure of a linear plasmid of the yeast Kluyveromyces lactis; Compact organization of the killer genome

Summary Some strains of the yeast Kluyveromyces lactis contain a pair of linear DNA plasmids, k1 and k2, 8.8 and 13.8 kilobase pairs long, respectively. Simultaneous presence of the two plasmids confer a killer phenotype on the cell by producing a toxin which blocks the growth of sensitive yeast species. Previous genetic studies have suggested that the toxin protein is coded by the k1 plasmid. We have now determined the total nucleotide sequence of k1 DNA. The genome is 8,874 base pairs in length. It contains four protein-coding reading frames, three transcribed from one strand and the fourth transcribed from the complementary strand and has terminal inverted repeats of 202 base pairs. Nuclease S1 mapping confirmed this arrangement and showed that these genes are transcribed. The terminal repeats and the four genes form an extremely compact genome, with some overlapping of genes. All four genes use highly biased codons, 86% of them having A or T at the wobble position, reminiscent of yeast mitochondrial genes. Three genes share a very similar 5 leader sequence. The nature of gene products is discussed in the light of what is known of the excreted toxin protein.     

The organization of repeating units in mitochondrial DNA from yeast petite mutants

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ^– petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.     

Amphimeric mitochondrial genomes of petite mutants of yeast. III. Generation by linking two secondary–structure-dependent illegitimate recombination events

The generation of amphimeric mitochondrial petite genomes of yeast can be explained by a process that links together two illegitimate recombination events, each involving a pair of short inverted repeats. Following “diagonal” double-strand breaks and inter-strand ligations at both possible stem-and-loop structures, a subgenomic single-stranded DNA circle can be excised. This circle comprises four building blocks organized in the so-called datA arrangement where d and t correspond, respectively, to the segments looped out by the upstream and the downstream pair of inverted repeats, a to the sequence separating the two loops, and A to the inverted duplication of segment a. Depending on the different possible “diagonal” recombinations at the inverted repeats, any of four isomeric circles can be excised, representing in its double-stranded form the nascent basic unit of an amphimeric mitochondrial petite genome of yeast. These isomeric basic units differ in the relative orientation of their sequences d and t (called D and T, respectively, when inverted), and are designated datA, DatA, daTA, and DaTA. Any one of these may be replicated to form the previously described regularly arrayed multimeric flip-flop genomes. Our new understanding of the amphimeric mitochondrial petite genomes of yeast emphasizes the role that topological features of DNA can play in mitochondrial genome dynamics. It also permits the re-interpretation of various observations reported in the literature. Some of them, including EtBr-mutagenesis in yeast, are discussed. Received: 13 July / 15 October 1998     

Control of hair follicle cell fate by underlying mesenchyme through a CSL–Wnt5a–FoxN1 regulatory axis

Epithelial–mesenchymal interactions are key to skin morphogenesis and homeostasis. We report that maintenance of the hair follicle keratinocyte cell fate is defective in mice with mesenchymal deletion of the CSL/RBP-Jκ gene, the effector of “canonical” Notch signaling. Hair follicle reconstitution assays demonstrate that this can be attributed to an intrinsic defect of dermal papilla cells. Similar consequences on hair follicle differentiation result from deletion of Wnt5a, a specific dermal papilla signature gene that we found to be under direct Notch/CSL control in these cells. Functional rescue experiments establish Wnt5a as an essential downstream mediator of Notch–CSL signaling, impinging on expression in the keratinocyte compartment of FoxN1, a gene with a key hair follicle regulatory function. Thus, Notch/CSL signaling plays a unique function in control of hair follicle differentiation by the underlying mesenchyme, with Wnt5a signaling and FoxN1 as mediators.     

Targeted next-generation sequencing reveals further genetic heterogeneity in axonal Charcot–Marie–Tooth neuropathy and a mutation in HSPB1

Charcot–Marie–Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype–phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches.     

Upregulation of Immunoglobulin‐related Genes in Cortical Sections from Multiple Sclerosis Patients

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Microarray‐based global gene expression profiling is a promising method, used to study potential genes involved in the pathogenesis of the disease. In the present study, we have examined global gene expression in normal‐appearing gray matter and gray matter lesions from the cortex of MS patients, and compared them with cortical gray matter samples from controls. We observed a massive upregulation of immunoglobulin (Ig)‐related genes in cortical sections of MS patients. Using immunohistochemistry, the activation of Ig genes seems to occur within plasma cells in the meninges. As synthesis of oligoclonal IgGs has been hypothesized to be caused by the activation of Epstein–Barr virus (EBV)‐infected B‐cells, we screened the brain samples for the presence of EBV by real‐time quantitative polymerase chain reaction (qPCR) and immunohistochemistry, but no evidence of active or latent EBV infection was detected. This study demonstrates that genes involved in the synthesis of Igs are upregulated in MS patients and that this activation is caused by a small number of meningeal plasma cells that are not infected by EBV. The findings indicate that the Ig‐producing B‐cells found in the cerebrospinal fluid (CSF) of MS patients could have meningeal origin.     

No evidence for intra-individual correlations between sleep-mediated declarative memory consolidation and slow-wave sleep

Study ObjectivesMemory consolidation benefits from a retention period filled with sleep. Several theoretical accounts assume that slow-wave sleep (SWS) contributes functionally to processes underlying the stabilization of declarative memories during sleep. However, reports on correlations between memory retention and the amount of SWS are mixed and typically rely on between-subject correlations and small sample sizes. Here we tested for the first time whether the amount of SWS during sleep predicts the effect of sleep on memory consolidation on an intra-individual level in a large sample.MethodsOne hundred and fifty-nine healthy participants came to the lab twice and took a 90 min nap in both sessions. Sleep-mediated memory benefits were tested using the paired associates word-learning task in both sessions.ResultsIn contrast to the theoretical prediction, intra-individual differences in sleep-mediated memory benefits did not significantly correlate with differences in SWS or SWA between the two naps. Also between subjects, the amount of SWS did not correlate with memory retention across the nap. However, subjective ratings of sleep quality were significantly associated with the amount of SWS.ConclusionOur results question the notion that the amount of SWS per se is functionally related to processes of memory consolidation during sleep. While our results do not exclude an important role of SWS for memory, they suggest that “more SWS” does not necessarily imply better memory consolidation.     

Constitutively active NF-κB triggers systemic TNFα-dependent inflammation and localized TNFα-independent inflammatory disease

NF-κB is well established as a key component of the inflammatory response. However, the precise mechanisms through which NF-κB activation contributes to inflammatory disease states remain poorly defined. To test the role of NF-κB in inflammation, we created a knock-in mouse that expresses a constitutively active form of NF-κB p65 dimers. These mice are born at normal Mendelian ratios, but display a progressive, systemic hyperinflammatory condition that results in severe runting and, typically, death 8–20 d after birth. Examination of homozygous knock-in mice demonstrates significant increases in proinflammatory cytokines and chemokines. Remarkably, crossing this strain with mice lacking TNF receptor 1 (TNFR1) leads to a complete rescue of the hyperinflammatory phenotype. However, upon aging, these rescued mice begin to display chronic keratitis accompanied by increased corneal expression of TNFα, IL-1β, and MMP-9, similar to that seen in human keratoconjunctivitis sicca (KCS) or “dry eyes.” Therefore, our results show that, while constitutively active NF-κB can trigger systemic inflammation, it does so indirectly, through increased TNF production. However, certain inflammatory disease states, such as keratitis or KCS, a condition that is seen in Sjogren''s syndrome, are dependent on NF-κB, but are independent of TNFR1 signaling.     

Is There a Weekly Pattern for Health Searches on Wikipedia and Is the Pattern Unique to Health Topics?

Background

Online health information–seeking behaviors have been reported to be more common at the beginning of the workweek. This behavior pattern has been interpreted as a kind of “healthy new start” or “fresh start” due to regrets or attempts to compensate for unhealthy behavior or poor choices made during the weekend. However, the observations regarding the most common health information–seeking day were based only on the analyses of users’ behaviors with websites on health or on online health-related searches. We wanted to confirm if this pattern could be found in searches of Wikipedia on health-related topics and also if this search pattern was unique to health-related topics or if it could represent a more general pattern of online information searching—which could be of relevance even beyond the health sector.

Objective

The aim was to examine the degree to which the search pattern described previously was specific to health-related information seeking or whether similar patterns could be found in other types of information-seeking behavior.

Methods

We extracted the number of searches performed on Wikipedia in the Norwegian language for 911 days for the most common sexually transmitted diseases (chlamydia, gonorrhea, herpes, human immunodeficiency virus [HIV], and acquired immune deficiency syndrome [AIDS]), other health-related topics (influenza, diabetes, and menopause), and 2 nonhealth-related topics (footballer Lionel Messi and pop singer Justin Bieber). The search dates were classified according to the day of the week and ANOVA tests were used to compare the average number of hits per day of the week.

Results

The ANOVA tests showed that the sexually transmitted disease queries had their highest peaks on Tuesdays (P<.001) and the fewest searches on Saturdays. The other health topics also showed a weekly pattern, with the highest peaks early in the week and lower numbers on Saturdays (P<.001). Footballer Lionel Messi had the highest mean number of hits on Tuesdays and Wednesdays, whereas pop singer Justin Bieber had the most hits on Tuesdays. Both these tracked search queries also showed significantly lower numbers on Saturdays (P<.001).

Conclusions

Our study supports prior studies finding an increase in health information searching at the beginning of the workweek. However, we also found a similar pattern for 2 randomly chosen nonhealth-related terms, which may suggest that the search pattern is not unique to health-related searches. The results are potentially relevant beyond the field of health and our preliminary findings need to be further explored in future studies involving a broader range of nonhealth-related searches.     

PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale

Peroxisome proliferator-activated receptor γ(PPARγ), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPARγ binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPARγ binding in mouse 3T3-L1 adipocytes. The consensus PPARγ/RXRα “DR-1”-binding motif was found at most of the sites, and ChIP for RXRα showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPARγ-binding sites, and genome-wide analysis of C/EBPα binding demonstrated that it localized to 3350 of the locations bound by PPARγ. Importantly, most genes induced in adipogenesis were bound by both PPARγ and C/EBPα, while very few were PPARγ-specific. C/EBPβ also plays a role at many of these genes, such that both C/EBPα and β are required along with PPARγ for robust adipocyte-specific gene expression. Thus, PPARγ and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.     

Deletions involving genes WHSC1 and LETM1 may be necessary,but are not sufficient to cause Wolf–Hirschhorn Syndrome

Wolf–Hirschhorn syndrome (WHS) is a complex genetic disorder caused by the loss of genomic material from the short arm of chromosome 4. Genotype–phenotype correlation studies indicated that the loss of genes within 4p16.3 is necessary for expression of the core features of the phenotype. Within this region, haploinsufficiency of the genes WHSC1 and LETM1 is thought to be a major contributor to the pathogenesis of WHS. We present clinical findings for three patients with relatively small (<400 kb) de novo interstitial deletions that overlap WHSC1 and LETM1. 3D facial analysis was performed for two of these patients. Based on our findings, we propose that hemizygosity of WHSC1 and LETM1 is associated with a clinical phenotype characterized by growth deficiency, feeding difficulties, and motor and speech delays. The deletion of additional genes nearby WHSC1 and LETM1 does not result in a marked increase in the severity of clinical features, arguing against their haploinsufficiency. The absence of seizures and typical WHS craniofacial findings in our cohort suggest that deletion of distinct or additional 4p16.3 genes is necessary for expression of these features. Altogether, these results show that although loss-of-function for WHSC1 and/or LETM1 contributes to some of the features of WHS, deletion of additional genes is required for the full expression of the phenotype, providing further support that WHS is a contiguous gene deletion disorder.     

Bidirectional fusion of the heart-forming fields in the developing chick embryo.

It is generally thought that the early pre-tubular chick heart is formed by fusion of the anterior or cephalic limits of the paired cardiogenic fields. However, this study shows that the heart fields initially fuse at their midpoint to form a transitory "butterfly"-shaped, cardiogenic structure. Fusion then progresses bi-directionally along the longitudinal axis in both cranial and caudal directions. Using in vivo labeling, we demonstrate that cells along the ventral fusion line are highly motile, crossing future primitive segments. We found that mesoderm cells migrated cephalically from the unfused tips of the anterior/cephalic wings into the head mesenchyme in the region that has been called the secondary heart field. Perturbing the anterior/cranial fusion results in formation of a bi-conal heart. A theoretical role of the ventral fusion line acting as a "heart organizer" and its role in cardia bifida is discussed.     

High frequency of copy number imbalances in Rubinstein–Taybi patients negative to CREBBP mutational analysis

Rubinstein–Taybi syndrome (RSTS) is a rare autosomal dominant disorder characterised by facial dysmorphisms, growth and psychomotor development delay, and skeletal defects. The known genetic causes are point mutations or deletions of the CREBBP (50–60%) and EP300 (5%) genes. To detect chromosomal rearrangements indicating novel positional candidate RSTS genes, we used a-CGH to study 26 patients fulfilling the diagnostic criteria for RSTS who were negative at fluorescence in situ hybridisation analyses of the CREBBP and EP300 regions, and direct sequencing analyses of the CREBBP gene. We found seven imbalances (27%): four de novo and three inherited rearrangements not reported among the copy number variants. A de novo 7p21.1 deletion of 500 kb included the TWIST1 gene, a suggested candidate for RSTS that is responsible for the Saethre–Chotzen syndrome, an entity that enters in differential diagnosis with RSTS. A similar issue of differential diagnosis was raised by a large 4.3 Mb 2q22.3q23.1 deletion encompassing ZEB2, the gene responsible for the Mowat–Wilson syndrome, whose signs may overlap with RSTS. Positional candidate genes could not be sought in the remaining pathogenetic imbalances, because of the size of the involved region (a 9 Mb 2q24.3q31.1 deletion) and/or the relative paucity of suitable genes (a 5 Mb 3p13p12.3 duplication). One of the inherited rearrangements, the 17q11.2 379Kb duplication, represents the reciprocal event of the deletion underlying an overgrowth syndrome, both being mediated by the NF1-REP-P1 and REP-P2 sub-duplicons. The contribution of this and the other detected CNVs to the clinical RSTS phenotype is difficult to assess.