Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome

The Proper Splicing of RNAi Factors Is Critical for Pericentric Heterochromatin Assembly in Fission Yeast

Heterochromatin preferentially assembles at repetitive DNA elements, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNAi machinery is required for heterochromatin assembly in a diverse range of organisms. In fission yeast, RNA splicing factors are also required for pericentric heterochromatin assembly, and a prevailing model is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, by screening the fission yeast deletion library, we discovered four novel splicing factors that are required for pericentric heterochromatin assembly. Sequencing total cellular RNAs from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors. Moreover, introducing cDNA versions of RNAi factors significantly restored pericentric heterochromatin in splicing mutants. We also found that mutations of splicing factors resulted in defective telomeric heterochromatin assembly and mis-splicing the mRNA of shelterin component Tpz1, and that replacement of tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus, proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres

Theory and practice of social norms interventions: eight common pitfalls.

BACKGROUND: Recently, Global Health practitioners, scholars, and donors have expressed increased interest in "changing social norms" as a strategy to promote health and well-being in low and mid-income countries (LMIC). Despite this burgeoning interest, the ability of practitioners to use social norm theory to inform health interventions varies widely. MAIN BODY: Here, we identify eight pitfalls that practitioners must avoid as they plan to integrate a social norms perspective in their interventions, as well as eight learnings. These learnings are: 1) Social norms and attitudes are different; 2) Social norms and attitudes can coincide; 3) Protective norms can offer important resources for achieving effective social improvement in people's health-related practices; 4) Harmful practices are sustained by a matrix of factors that need to be understood in their interactions; 5) The prevalence of a norm is not necessarily a sign of its strength; 6) Social norms can exert both direct and indirect influence; 7) Publicising the prevalence of a harmful practice can make things worse; 8) People-led social norm change is both the right and the smart thing to do. CONCLUSIONS: As the understanding of how norms evolve in LMIC advances, practitioners will develop greater understanding of what works to help people lead change in harmful norms within their contexts. Awareness of these pitfalls has helped several of them increase the effectiveness of their interventions addressing social norms in the field. We are confident that others will benefit from these reflections as well

The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

The Roles of Splicing and H2A.Z in Chromatin Assembly

Eukaryotic nuclear DNA is folded with histone and non-histone proteins into chromatin, a nucleoprotein structure regulated by histone post-translational modifications and substitution with histone variants. Chromatin mediates processes such as DNA damage repair, cell differentiation, gene silencing, and centromere specification. Mistaken inheritance of chromatin-mediated gene silencing, for instance, can cause both aberrant development and cancer. Gene silencing at pericentromeres and centromeres, which can be attained through obstruction of transcription as well as through recruitment of specific RNA-degrading proteins, is essential for centromere specification. However, the molecular mechanisms of these processes are not yet thoroughly understood, and therefore they will be the focus of this thesis. A structure termed heterochromatin, for which the essential hallmark is histone H3 lysine 9 methylation (H3K9me), preferentially assembles at repetitive DNA such as pericentric regions, playing roles in transcriptional silencing, recombination suppression, and chromosome segregation. The RNA interference (RNAi) machinery is required for heterochromatin assembly over DNA repeats in diverse organisms by targeting histone-modifying activities. Surprisingly, RNA splicing factors are also required for this process. A widely-held model derived from studies in fission yeast is that splicing factors provide a platform for siRNA generation independently of their splicing activity. Here, we discovered the requirement of four non-essential splicing factors for pericentric heterochromatin assembly, allowing us to more clearly address the role of splicing in heterochromatin assembly. Sequencing total cellular RNA from the strongest of these mutants, cwf14Δ, showed intron retention in mRNAs of several RNAi factors, which correspond to strong reduction in levels of a central RNAi protein, Argonaute. Moreover, introducing cDNA versions of RNAi factors significantly restores pericentric heterochromatin in splicing mutants. We also found that mutation of splicing factors affects telomeric heterochromatin, and replacement of mis-spliced factor tpz1+ with its cDNA partially rescued heterochromatin defects at telomeres in splicing mutants. Thus proper splicing of RNAi and shelterin factors contributes to heterochromatin assembly at pericentric regions and telomeres. In addition to post-translational modifications, chromatin silencing can be regulated by histone variants such as H2A.Z. The incorporation of H2A.Z into chromatin regulates chromatin structure and gene expression. The Swr1 chromatin remodeling complex deposits H2A.Z in budding yeast and mammals. Here we characterize a novel component of the fission yeast Swr1 complex, Msc1, which is a Jumonji domain protein frequently associated with histone demethylation. We found that Msc1 is required for Swr1-mediated incorporation of H2A.Z into chromatin at gene promoters. We demonstrated that H2A.Z is required for the expression of CENP-C, which in turn regulates centromere silencing and chromosome segregation. Together, these results show that chromatin silencing at pericentromeres and centromeres is mediated by splicing factors and H2A.Z, respectively, to promote proper regulation of other chromatin factors, thus ensuring faithful chromosome segregation

Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly

The RNAi pathway is required for heterochromatin assembly at repetitive DNA elements in diverse organisms. In fission yeast, loss of RNAi causes pericentric heterochromatin defects, compromising gene silencing and chromosome segregation. Here we show that deletion of telomere shelterin components restores pericentric heterochromatin and its functions in RNAi mutants. We further isolated a separation-of-function mutant of Poz1 and revealed that defective telomere silencing, but not telomere length control, is critical for bypassing RNAi. Further analyses demonstrated that compromising shelterin-mediated heterochromatin assembly in RNAi mutants releases heterochromatin protein Swi6, which is redistributed to pericentric regions through RNAi-independent heterochromatin assembly pathways. Given the high mobility of Swi6 protein and that increased levels of Swi6 facilitates heterochromatin spreading as well as ectopic heterochromatin assembly, our results suggest that constitutive heterochromatin domains use multiple pathways to form high-affinity platforms to restrain Swi6, thus limiting its availability and avoiding promiscuous heterochromatin formation

The Food and Drug Administration Office of Women\u27s Health: Impact of science on regulatory policy

In 1994, the Food and Drug Administration Office of Women\u27s Health (FDA-OWH) was created to provide leadership and policy direction for the Agency regarding issues of women\u27s health. Within its first year, the FDA-OWH established a science program for women\u27s health research, promoting the development of sound policy and regulation. In a little over a decade, the program has provided approximately $14 million to fund more than 100 women\u27s health research studies covering a broad range of health topics affecting women across their lifespan. Some studies, such as those elucidating drug effects on QT prolongation in women and drug-dietary supplement interaction, have had significant influence on regulatory decisions. Other studies have provided sound scientific data on sex and gender differences supporting FDA guidelines to protect women\u27s health. This paper describes the science program at the FDA-OWH, providing examples of how funded research impacts regulatory policy. © Mary Ann Liebert, Inc

Hydroxyurea (HU) treatment enhances centromeric PEV.

<p><b>(A)</b> HU promotes switching in expression states of <i>cnt2</i>::<i>ade6</i>⁺ in a dosage-dependent manner. Cell suspensions of a red (upper panel) or a white (lower panel) colony are grown in liquid media with HU as labeled for 24 hours, washed and spread onto YE+4S (low adenine) plates, with >500 cells/plate. The red or white colonies are counted at 5th day when incubated at 25°C. Each experiment has been performed by at least triple biological repeats. Each biological repeat has its unique composition of red/white cell ratio in the initial culture. One biological experiment result is shown here. The error bars are 1 SD of percentages for four plates. <i>p</i> values are calculated by Fisher exact test. <b>(B)</b> The red colony exhibits enhanced sectoring pattern upon HU treatment. Cells derived from the above red colony (before HU treatment) are planted evenly at one cell/cm² by microscopic manipulation on YE+4S plates containing HU as labeled, incubated at 25°C for eight days. Two representative colonies for each are shown. The number shown here means the number of red colonies in the total colonies. <b>(C)</b> Elevated frequencies of <i>cnt2</i>::<i>ade6</i>^<i>+</i> red-to-white switching measured by pedigree analysis in wild type cells with HU treatment and <i>cdc22-3</i> mutants. Pedigree analyses are performed on “hybrid” plates for measuring switching rate of HU treated cells, each with a drug-free zone and a drug-containing zone. For each pedigree, three cell generations are tracked at the zone containing HU as labeled. The eight progeny cells are then moved to the drug-free zone, incubated at 25^°C for five days. For each experimental condition (wild type with or without HU treatment and <i>cdc22-3</i> mutant), colony color switching events are scored in 400–1000 cell division events in total. The error bars are 1 SD of percentages for four independent experiments. <b>(D)</b> Colonies maintain the wild type level of sectoring after short-term HU treatment. Cell suspensions of a red colony in (A) grown under 2mM HU is washed and planted evenly at 1 cell/cm² in YE4S media, and incubated at 25°C for eight days.</p

HU affects PEV at heterochromatin region.

<p><b>(A)</b> HU affects PEV within the mating type locus. Diagram illustrates the location of <i>ade6</i>⁺ insertion at the mating type locus, between the centromere-proximal boundary and the <i>mat2-P</i> gene (strain AP144—<i>mat_L(BglII)</i>::<i>ade6</i>⁺). Dark green box indicates a normal silencing domain. <b>(D)</b> HU treatment affects PEV induced by <i>cenH</i> at an ectopic site. Diagram illustrates the genetic construct of the strain (LW75- <i>cenH- ade6</i>^<i>+</i>). <i>cenH</i> and <i>ade6</i>^<i>+</i>are inserted in the <i>ura4</i>^<i>+</i> locus, replacing the majority of the <i>ura4</i>^<i>+</i> ORF. <b>(B-C, E-F)</b> For each strain, cell suspensions of a predominantly white colony (B, E) and red colony (C, F) are treated with HU as labeled for 24 hours, washed and plated onto YE+4S solid media, and incubated at 25^°C for 5 days. The red and white colonies are counted respectively, with 300–400 total/per plate. The error bars are 1 SD of percentages for four plates. <i>p</i> values are calculated by Fisher exact test.</p