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

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

Introducing cDNAs of RNAi factors rescues pericentric heterochromatin defects of <i>cwf14Δ</i> cells.

<p>(A) Serial dilution analyses to measure the expression of <i>otr::ura4⁺</i> and sensitivity to TBZ. (B) ChIP analyses of H3K9me2 and Swi6 levels at pericentric <i>dh</i> repeats and <i>otr::ura4⁺</i>, normalized to an <i>act1</i> promoter fragment. Error bars represent standard deviation of three experiments.</p

Splicing factors regulate the proper splicing of shelterin component Tpz1.

<p>(A) RT-PCR analyses of RNA with primers flanking introns. (B, E) Serial dilution analyses to measure reporter gene expression. (C) ChIP analyses of H3K9me2 levels, normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments. (D, F) qRT-PCR analyses of transcripts derived from <i>tlh1</i>, normalized to <i>act1</i> gene. Wild type was set to 1. Error bars represent standard deviation of three experiments.</p

Cwf14 associates with the spliceosome.

<p>(A) Tagged versions of Cwf14 are functional as indicated by serial dilution analysis. (B) Imaging of cells expressing Cwf14-GFP, with DAPI to stain nucleus. Scale bar, 10 µm. (C) Western blot analysis of Cwf14-myc expression. (D) Mass spectrometry analyses of purified Cwf14-myc complex. Factors were binned into annotated subcomplexes.</p

Cwf14 is required for pericentric heterochromatin assembly through the RNAi pathway.

<p>(A, E, F, and G) Serial dilution analyses to measure the expression of reporter genes and sensitivity to TBZ. (B and C) ChIP analyses of Pol II (Rpb1), H3K9me2, and Swi6 levels at pericentric <i>dh</i> repeats and <i>otr::ura4⁺</i>. Pol II ChIP was normalized to <i>act1</i> gene, and H3K9me2 and Swi6 ChIP was normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments. (D) Northern blot analyses of siRNAs derived from pericentric <i>dh</i> repeats.</p

Cwf14 is not required for CLRC activity.

<p>(A) Schematic diagram of the <i>ura4::3xgbs-ade6⁺</i> reporter. (B) Left, serial dilution analysis to measure reporter gene expression. Right, ChIP analysis of H3K9me2 levels at the reporter, normalized to <i>act1</i> promoter. Error bars represent standard deviation of three experiments.</p