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Abstract Background DNA microarray technology allows the analysis of genome structure and dynamics at genome-wide scale. Expression microarrays (EMA) contain probes for annotated open reading frames (ORF) and are widely used for the analysis of differential gene expression. By contrast, tiling microarrays (TMA) have a much higher probe density and provide unbiased genome-wide coverage. The purpose of this study was to develop a protocol to exploit the high resolution of TMAs for quantitative measurement of DNA strand-specific differential expression of annotated and non-annotated transcripts. Results We extensively filtered probes present in Affymetrix Genechip Yeast Genome 2.0 expression and GeneChip S. pombe 1.0FR tiling microarrays to generate custom Chip Description Files (CDF) in order to compare their efficiency. We experimentally tested the potential of our approach by measuring the differential expression of 4904 genes in the yeast Schizosaccharomyces pombe growing under conditions of oxidative stress. The results showed a Pearson correlation coefficient of 0.943 between both platforms, indicating that TMAs are as reliable as EMAs for quantitative expression analysis. A significant advantage of TMAs over EMAs is the possibility of detecting non-annotated transcripts generated only under specific physiological conditions. To take full advantage of this property, we have used a target-labelling protocol that preserves the original polarity of the transcripts and, therefore, allows the strand-specific differential expression of non-annotated transcripts to be determined. By using a segmentation algorithm prior to generating the corresponding custom CDFs, we identified and quantitatively measured the expression of 510 transcripts longer than 180 nucleotides and not overlapping previously annotated ORFs that were differentially expressed at least 2-fold under oxidative stress. Conclusions We show that the information derived from TMA hybridization can be processed simultaneously for high-resolution qualitative and quantitative analysis of the differential expression of well-characterized genes and of previously non-annotated and antisense transcripts. The consistency of the performance of TMA, their genome-wide coverage and adaptability to updated genome annotations, and the possibility of measuring strand-specific differential expression makes them a tool of choice for the analysis of gene expression in any organism for which TMA platforms are available.

The current study examines cohort (i.e., Generation X, Baby Boomers, and Silent Generation) and gender differences in the stability of Mexican-origin family caregivers over time. The data comes from Mexican-origin caregivers to community-dwelling older adults living in the west and southwestern United States from the Caregiving Supplement to the Hispanic Established Populations for the Epidemiologic Study of the Elderly (2010/11-2016). Multinomial logistic regressions with interaction and predicted probabilities demonstrate that female caregivers are more consistent in their caregiving role over male caregivers in every cohort over the two time points (RRR = 0.67; 95% CI = [0.01-0.38]). Among men, predicted probabilities indicate more caregivers from Baby Boomer (.17) and Generation X (0.12) cohorts remain in the caregiver role compared to Silent Generation (.07). The findings suggest that gendered expectations may be changing as male caregivers increase in their provision of family care. Future research should consider generational diversity in Hispanic caregiving experience., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The prevalence of prenatal tobacco exposure remains high in many countries, particularly in southern Europe. The aims of this study were to estimate the prevalence of smoking among pregnant women in a southern Spanish city (Seville) and to identify the associated sociodemographic and obstetric characteristics. In a descriptive, cross-sectional study, a random sample of pregnant women who were scheduled to undergo a morphology scan at their public referral hospital in their 20th week of gestation were interviewed in person. At the start of pregnancy, 38.2% of the pregnant women were smokers. In the twentieth week, 19.1% continued to smoke, and the same percentage had quit. The prevalence of smoking in pregnant women was higher among those with a low level of education (60% among pregnant women with no studies and 30.4% in those with primary education) and among those who had had abortions (38.5%). Pregnant smokers with obesity were the least likely to have given up smoking during pregnancy. Women with a lower educational level should be a prime target for cross-sectoral interventions aimed at preventing prenatal tobacco exposure. Implementation of support measures for providing effective clinical advice in preconception and prenatal care regarding healthy lifestyles is particularly needed.

Background and Objectives: To examine the role of probable dementia on changes in living arrangements and mortality among very old Mexicans and Mexican Americans in 2 different nations., Research Design and Methods: We employ the Hispanic Established Population for the Epidemiologic Study of the Elderly and the Mexican Health and Aging Study, 2 comparable longitudinal data sets, to identify predictors of changes in living arrangements using multinomial logistic regression, controlling for cognitive status, demographic characteristics, and resources., Results: In Mexico, women with dementia who lived alone at baseline were more likely to become part of an extended family household than men with similar levels of cognitive impairment. A similar pattern emerges for the oldest Mexican-American women. Spousal loss increases the likelihood of living alone for women in the United States regardless of dementia. Although dementia elevates the risk of mortality for men living alone in the United States, in both countries, women in their 90s who lived alone with dementia had a lower risk of mortality relative to men., Discussion and Implications: Longer life spans increase the risk of living alone with dementia in both countries, especially for women. Older individuals in both countries face financial hardships. Mexicans have limited formal options in dementia care. Mexican Americans with dementia continue to live alone despite low income although, unlike the Mexicans, they have access to Medicaid long-term care. For Mexico and the United States, the growing number of older individuals with dementia represents a growing public health concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the symmetrical structure of nucleosomes, in vitro studies have shown that transcription proceeds with different efficiency depending on the orientation of the DNA sequence around them. However, it is unclear whether this functional asymmetry is present in vivo and whether it could regulate transcriptional directionality. Here, we report that the proximal and distal halves of nucleosomal DNA contribute differentially to nucleosome stability in the genome. In +1 nucleosomes, this asymmetry facilitates or hinders transcription depending on the orientation of its underlying DNA, and this difference is associated with an asymmetrical interaction between DNA and histones. These properties are encoded in the DNA signature of +1 nucleosomes, since its incorporation in the two orientations into downstream nucleosomes renders them asymmetrically accessible to MNase and inverts the balance between sense and antisense transcription. Altogether, our results show that nucleosomal DNA endows nucleosomes with asymmetrical properties that modulate the directionality of transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: None.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs., Competing Interests: Declaration of interests J.D.B. is a Founder and Director of CDI Labs, Inc.; a Founder of and consultant to Neochromosome, Inc.; a Founder, SAB member of, and consultant to ReOpen Diagnostics, LLC; and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The stability of the genome is occasionally challenged by the formation of DNA-RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA-RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On the one hand, Rtt109 prevents DNA-RNA hybridization by the acetylation of histone H3 lysines 14 and 23 and, on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA-RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Pregnant women must maintain or acquire healthy habits during pregnancy to protect both their own health and their child's. Such habits include an adequate eating pattern along with good adherence to the intake of certain supplements, practice of moderate physical activity and avoiding the consumption of toxic products such as tobacco and alcohol. The objective of this study is to assess the interrelation between such habits and their association with sociodemographic variables. To such end, a cross-sectional study was conducted with a representative sample of pregnant women who attended the scheduled morphology echography consultation at the 20th gestational week in their reference public hospital in the city of Seville (Spain). Results: Younger pregnant women and with lower educational levels are the ones that present the worst eating habits and the highest smoking rate. Pregnant women with lower educational levels are the least active. Non-smoking pregnant women present better eating habits than those who smoke. Pregnant women with lower educational levels are those who accumulate more unhealthy habits during pregnancy. This should be taken into account when planning the health care provided to pregnant women and in public health intersectoral policies.

Homologous recombination is essential to maintain genome stability in response to DNA damage. Here, we have used genome-wide sequencing to quantitatively analyze at nucleotide resolution the dynamics of DNA end resection, re-synthesis, and gene conversion at a double-strand break. Resection initiates asymmetrically in an MRX-independent manner before proceeding steadily in both directions. Sgs1, Exo1, Rad51, and Srs2 differently regulate the rate and symmetry of early and late resection. Exo1 also ensures the coexistence of resection and re-synthesis, while Srs2 guarantees a constant and symmetrical DNA re-polymerization. Gene conversion is MMR independent, spans only a minor fraction of the resected region, and its unidirectionality depends on Srs2. Finally, these repair factors prevent the development of alterations remote from the DNA lesion, such as subtelomeric instability, duplication of genomic regions, and over-replication of Ty elements. Altogether, this approach allows a quantitative analysis and a direct genome-wide visualization of DNA repair by homologous recombination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Prenatal care, understood as routine care provided during pregnancy, should be based on basic pillars, such as taking into account the sociocultural context in which such care is offered, ensuring that the service is appropriate, accessible and of high quality, and also with care personalized. According to World Health Organization, the minimum number of recommended visits during pregnancy should be eight, with the different professionals involved in the process, in addition to preparing for childbirth or maternal education. Since the pandemic erupted due to SARS-CoV-2, many of the usual social health services have had to adapt to provide safety and prevent infection, a priority in vulnerable groups where pregnant women are found. Professionals have had to adapt to telematics care, thus attending consultations to reduce the mobility of pregnant women to health centers, thus avoiding unnecessary risks. Therefore, all this situation has opened a virtual field of work that, although previously carried out in different areas, now more than ever acquires special relevance and for which professional training is necessary, as a complement to face-to-face appointments.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.

Motivation: The Burrows-Wheeler transform (BWT) is widely used for the fast alignment of high-throughput sequence data. This method also has potential applications in other areas of bioinformatics, and it can be specially useful for the fast searching of patterns on coverage data from different sources., Results: We present a nucleosome pattern search method that converts levels of nucleosomal occupancy to a sequence-like format to which BWT searches can be applied. The method is embedded in a nucleosome map browser, 'Nucleosee', an interactive visual tool specifically designed to enhance BWT searches, giving them context and making them suitable for visual discourse analysis of the results. The proposed method is fast, flexible and sufficiently generic for the exploration of data in a broad and interactive way., Availability and Implementation: The proposed algorithm and visual browser are available for testing at http://cpg3.der.usal.es/nucleosee. The source code and installation packages are also available at https://github.com/rodrigoSantamaria/nucleosee., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study DNA metabolism, in which the DNA replication and repair mechanisms are evolutionarily conserved. In this introduction we describe a range of methods commonly used to study aspects of DNA metabolism in fission yeast, focusing on approaches used for the analysis of genome stability, DNA replication, and DNA repair. We describe the use of a minichromosome, Ch 16 , for monitoring different aspects of genome stability. We introduce two-dimensional gel electrophoresis and immunofluorescent visualization of combed DNA molecules for the analysis of DNA replication. Further, we introduce a pulsed field gel electrophoresis (PFGE) assay to physically monitor chromosome integrity, which can be used in conjunction with a DNA double-strand break (DSB) repair assay to genetically quantitate different DSB repair and misrepair outcomes, including gross chromosomal rearrangements, in fission yeast., (© 2018 Cold Spring Harbor Laboratory Press.)

Two-dimensional gel electrophoresis allows direct detection of DNA replication and recombination intermediates in preparations of total genomic DNA. This technique is widely used to identify replication origins in the yeast genome and is based on the different mobility in agarose gels of linear and branched DNA molecules depending on their mass and structure. During the first dimension, low-voltage and a low-percentage agarose gel favors separation of the molecules by their mass and minimizes the effect of their structure. In contrast, during the second dimension, a higher voltage, a higher percentage agarose gel, and the presence of ethidium bromide significantly delays the migration of branched structures relative to linear molecules of the same mass. This technique is appropriate for the detection of replication initiation and, therefore, an active origin of replication, within regions of approximately 2.5-5.5 kb. Consequently, it is not well suited for genome-wide replication analyses., (© 2018 Cold Spring Harbor Laboratory Press.)

The discovery of CpG islands (CGIs) and the study of their structure and properties run parallel to the development of molecular biology in the last two decades of the twentieth century and to the development of high-throughput genomic technologies at the turn of the millennium. First identified as discrete G + C-rich regions of unmethylated DNA in several vertebrates, CGIs were soon found to display additional distinctive chromatin features from the rest of the genome in terms of accessibility and of the epigenetic modifications of their histones. These features, together with their colocalization with promoters and with origins of DNA replication in mammals, highlighted their relevance in the regulation of genomic processes. Recent approaches have shown with unprecedented detail the dynamics and diversity of the epigenetic landscape of CGIs during normal development and under pathological conditions. Also, comparative analyses across species have started revealing how CGIs evolve and contribute to the evolution of the vertebrate genome.

Nucleosomes are the basic units of chromatin. They compact the genome inside the nucleus and regulate the access of proteins to DNA. In the yeast genome, most nucleosomes occupy well-defined positions, which are maintained under many different physiological situations and genetic backgrounds. Although several short sequence elements have been described that favor or reduce the affinity between histones and DNA, the extent to which the DNA sequence affects nucleosome positioning in the genomic context remains unclear. Recent analyses indicate that the base composition pattern of mononucleosomal DNA differs among species, and that the same sequence elements have a different impact on nucleosome positioning in different genomes despite the high level of phylogenetic conservation of histones. These studies have also shown that the DNA sequence contributes to nucleosome positioning to the point that it is possible to design synthetic DNA molecules capable of generating regular and species-specific nucleosomal patterns in vivo.

This corrects the article DOI: 10.1038/nature04585.

The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodelling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation requires, at least partially, RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits do not define alternate versions of the complex.

In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization., (© 2016 González et al.; Published by Cold Spring Harbor Laboratory Press.)

In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis. In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation (Materne et al., 2015) but the regulatory steps remain poorly understood. Here we report a genetic screen that highlighted H2B deubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2Bubi at the promoter where it represses expression, and over the transcribed region where it sustains it. By promoting H3K4 methylation at the promoter, H2Bubi initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR (nucleosome depleted region) formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.

The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions., (© 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.)

Within the assistance and support to coping with perinatal death, it must be considered that there is a group of women whose process has some features that give specific connotations. We talked about when the perinatal loss occurs due to a maternal decision to the presence of a fetal malformation. These cases today, thanks to advances in the techniques of control fetal development, are not uncommon. In their assistance, healthcare professionals should be aware that they often present a great sense of guilt and ambivalence between well-made decision and the hardness of having to come to it. A case of a pregnant woman undergoing a fetal fetolisis and care plan developed in her assistance for the induction of labor, delivery and immediate postpartum period is presented. This plan includes the problems of collaboration and the independent problems that are formulated according to the NANDA, NOC and NIC taxonomies. The implication for practice after studying this case leads to the duty to equally address the coping with a stillbirth, whether it was spontaneous or had it been determined by fetal malformation completion, giving parents the ability to view and contact with their child., (Copyright © 2015 Elsevier España, S.L.U. All rights reserved.)

Nucleosomes contribute to compacting the genome into the nucleus and regulate the physical access of regulatory proteins to DNA either directly or through the epigenetic modifications of the histone tails. Precise mapping of nucleosome positioning across the genome is, therefore, essential to understanding the genome regulation. In recent years, several experimental protocols have been developed for this purpose that include the enzymatic digestion, chemical cleavage or immunoprecipitation of chromatin followed by next-generation sequencing of the resulting DNA fragments. Here, we compare the performance and resolution of these methods from the initial biochemical steps through the alignment of the millions of short-sequence reads to a reference genome to the final computational analysis to generate genome-wide maps of nucleosome occupancy. Because of the lack of a unified protocol to process data sets obtained through the different approaches, we have developed a new computational tool (NUCwave), which facilitates their analysis, comparison and assessment and will enable researchers to choose the most suitable method for any particular purpose. NUCwave is freely available at http://nucleosome.usal.es/nucwave along with a step-by-step protocol for its use., (© The Author 2014. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.)

The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.

Nucleosomes are the basic structural units of chromatin. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under a wide range of physiological conditions. In this work, we have searched for sequence determinants associated with positioned nucleosomes in four species of fission and budding yeasts. We show that mononucleosomal DNA follows a highly structured base composition pattern, which differs among species despite the high degree of histone conservation. These nucleosomal signatures are present in transcribed and non-transcribed regions across the genome. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA, and they also determine a periodicity in the average distribution of amino acids along the proteins. These results establish a direct and species-specific connection between the position of each codon around the histone octamer and protein composition.

PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of β-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate β-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of β-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of β-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Nuclear RNAi is an important regulator of transcription and epigenetic modification, but the underlying mechanisms remain elusive. Using a genome-wide approach in the fission yeast S. pombe, we have found that Dcr1, but not other components of the canonical RNAi pathway, promotes the release of Pol II from the 3? end of highly transcribed genes, and, surprisingly, from antisense transcription of rRNA and tRNA genes, which are normally transcribed by Pol I and Pol III. These Dcr1-terminated loci correspond to sites of replication stress and DNA damage, likely resulting from transcription-replication collisions. At the rDNA loci, release of Pol II facilitates DNA replication and prevents homologous recombination, which would otherwise lead to loss of rDNA repeats especially during meiosis. Our results reveal a novel role for Dcr1-mediated transcription termination in genome maintenance and may account for widespread regulation of genome stability by nuclear RNAi in higher eukaryotes.

Background: Eukaryotic genomes are replicated during S phase according to a temporal program. Several determinants control the timing of origin firing, including the chromatin environment and epigenetic modifications. However, how chromatin structure influences the timing of the activation of specific origins is still poorly understood., Results: By performing high-resolution analysis of genome-wide nucleosome positioning we have identified different chromatin architectures at early and late replication origins. These different patterns are already established in G1 and are tightly correlated with the organization of adjacent transcription units. Moreover, specific early and late nucleosomal patterns are fixed robustly, even in rpd3 mutants in which histone acetylation and origin timing have been significantly altered. Nevertheless, higher histone acetylation levels correlate with the local modulation of chromatin structure, leading to increased origin accessibility. In addition, we conducted parallel analyses of replication and nucleosome dynamics that revealed that chromatin structure at origins is modulated during origin activation., Conclusions: Our results show that early and late replication origins present distinctive nucleosomal configurations, which are preferentially associated to different genomic regions. Our data also reveal that origin structure is dynamic and can be locally modulated by histone deacetylation, as well as by origin activation. These data offer novel insight into the contribution of chromatin structure to origin selection and firing in budding yeast.

In the "Replicon Theory", Jacob, Brenner and Cuzin proposed the existence of replicators and initiators as the two major actors in DNA replication. Over the years, many protein components of initiators have been shown to be conserved in different organisms during evolution. By contrast, replicator DNA sequences (often referred to as replication origins) have diverged beyond possible comparison between eukaryotic genomes. Replication origins in the fission yeast Schizosaccharomyces pombe are made up of A+T-rich sequences that do not share any consensus elements. The information encoded in these replicators is interpreted by the Orc4 subunit of the ORC (origin recognition complex), which is unique among eukaryotes in that it contains a large domain harboring nine AT-hook subdomains that target ORC to a great variety of A+T-rich sequences along the chromosomes. Recently, the genomes of other Schizosaccharomyces species have been sequenced and the regions encompassing their replication origins have been identified. DNA sequence analysis and comparison of the organization of their Orc4 proteins have revealed species-specific differences that contribute to our understanding of how the specification of replication origins has evolved during the phylogenetic divergence of fission yeasts., (© 2013.)

Background: Nucleosomes facilitate the packaging of the eukaryotic genome and modulate the access of regulators to DNA. A detailed description of the nucleosomal organization under different transcriptional programmes is essential to understand their contribution to genomic regulation., Results: To visualize the dynamics of individual nucleosomes under different transcriptional programmes we have generated high-resolution nucleosomal maps in Schizosaccharomyces pombe. We show that 98.5% of the genome remains almost invariable during mitosis and meiosis while remodelling is limited to approximately 1100 nucleosomes in the promoters of a subset of meiotic genes. These inducible nucleosome-depleted regions (NDR) and also those constitutively present in the genome overlap precisely with clusters of binding sites for transcription factors (TF) specific for meiosis and for different functional classes of genes, respectively. Deletion of two TFs affects only a small fraction of all the NDRs to which they bind in vivo, indicating that TFs collectively contribute to NDR maintenance., Conclusions: Our results show that the nucleosomal profile in S. pombe is largely maintained under different physiological conditions and patterns of gene expression. This relatively constant landscape favours the concentration of regulators in constitutive and inducible NDRs. The combinatorial analysis of binding motifs in this discrete fraction of the genome will facilitate the definition of the transcriptional regulatory networks.

In Schizosaccharomyces pombe, DNA replication origins (ORIs) and meiotic recombination hotspots lack consensus sequences and show a bias towards mapping to large intergenic regions (IGRs). To explore whether this preference depended on underlying chromatin features, we have generated genome-wide nucleosome profiles during mitosis and meiosis. We have found that meiotic double-strand break sites (DSBs) colocalize with nucleosome-depleted regions (NDRs) and that large IGRs include clusters of NDRs that overlap with almost half of all DSBs. By contrast, ORIs do not colocalize with NDRs and they are regulated independently of DSBs. Physical relocation of NDRs at ectopic loci or modification of their genomic distribution during meiosis was paralleled by the generation of new DSB sites. Over 80% of all meiotic DSBs colocalize with NDRs that are also present during mitosis, indicating that the recombination pattern is largely dependent on constitutive properties of the genome and, to a lesser extent, on the transcriptional profile during meiosis. The organization of ORIs and of DSBs regions in S. pombe reveals similarities and differences relative to Saccharomyces cerevisiae.

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification during the DNA replication phase of the cell cycle. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes.

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25-30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals.