The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.

Histone H2B ubiquitylation promotes activity of the intact Set1 histone methyltransferase complex in fission yeast.

The methylation of histone H3 at lysine 4 (H3K4me) is critical for the formation of transcriptionally active chromatin in eukaryotes. In yeast, Drosophila, and some human cell lines, H3K4me is globally stimulated by the monoubiquitylation of histone H2B (H2Bub1), another histone modification associated with transcription. The mechanism of this "trans-histone" modification pathway remains uncertain, and studies carried out in different experimental systems have suggested that H2Bub1 could either influence the subunit composition of methyltransferase complexes or directly stimulate methyltransferase activity. We have reconstituted this pathway in vitro using the native H3K4-specific methyltransferase complex Set1C purified from the fission yeast Schizosaccharomyces pombe and chromatin substrates that contain semisynthetic H2Bub1. We found that the activity of S. pombe Set1C toward nucleosomal histone H3 is directly enhanced by H2Bub1 in vitro. Importantly, Set1C purified from cells lacking H2Bub1 retained activity on free histone substrates, suggesting that Set1C remains intact in the absence of H2Bub1. Chromatin immunoprecipitation assays revealed a defect in recruitment of intact Set1C to transcribed chromatin in H2Bub1-deficient mutants. Our data argue that trans-histone crosstalk in S. pombe involves direct enhancement of Set1C methyltransferase activity by H2Bub1 and suggest that this represents a conserved aspect of H2Bub1-H3K4me crosstalk in eukaryotes.

The Glass Menagerie: diatoms for novel applications in nanotechnology.

Diatoms are unicellular, eukaryotic, photosynthetic algae that are found in aquatic environments. Diatoms have enormous ecological importance on this planet and display a diversity of patterns and structures at the nano- to millimetre scale. Diatom nanotechnology, a new interdisciplinary area, has spawned collaborations in biology, biochemistry, biotechnology, physics, chemistry, material science and engineering. We survey diatom nanotechnology since 2005, emphasizing recent advances in diatom biomineralization, biophotonics, photoluminescence, microfluidics, compustat domestication, multiscale porosity, silica sequestering of proteins, detection of trace gases, controlled drug delivery and computer design. Diatoms might become the first organisms for which the gap in our knowledge of the relationship between genotype and phenotype is closed.

Coital status and perceptions about sexual abstinence refusal skills.

PURPOSE: To compare perceptions and use of sexual refusal skills. METHODS: A voluntary, anonymous sample of ninth and tenth grade students (N = 2256) reported their perceptions about and use of sexual refusal skills that had been taught in school. Forty-four percent of the students classified themselves as virgins, 18% reported one sexual partner, and 38% reported multiple sex partners. Coital status groups were compared on measures addressing refusal skills using Chi-square tests. RESULTS: Approximately one-third of the students reported using refusal skills either a lot or a little, with virgins more likely to indicate that they used refusal skills a lot. Virgins also were least likely to say that they should have used the refusal skills or that they didn't feel like using refusal skills. Furthermore, virgins were most likely to state that they didn't need to use refusal skills. One partner and multiple partner groups were more likely to indicate that they had not been taught refusal skills. CONCLUSIONS: The majority of students did not use the refusal skills taught to them in school; however, all coital status groups used the refusal skills in some capacity. Differences between the virgin and sexually experienced groups present challenges for instructors and curriculum developers. Some of the commonly held assumptions about refusal skill curricula may not be true.

Scales measuring psychosocial antecedents of coital initiation among adolescents in a rural southern state.

The psychometric properties of psychosexual scales designed to examine intentions toward sexual intercourse were examined. Participants from 22 schools in central Alabama provided data on demographics, knowledge, attitudes, beliefs, and intentions toward sexual intercourse. Theoretical constructs from Theory of Reasoned Action and Social Cognitive Theory guided the development of items assessing psychosocial aspects of sexual intercourse. Findings indicated that approximately half of the conceptual items (13 items) loaded on four factors. The four scales were (a) Coital Intentions, (b) Attitudes toward Negative Sexual Outcomes, (c) Social Norms toward Premature Sex, and (d) Self-efficacy of Sexual Refusal Skills. These factors showed adequate independence and explained 46% of the variance in the data. Cronbach alpha ranged from .69 to .78. Scales show acceptable evidence for evaluation; however, additional research from other geographical districts and other ethnic groups is needed to confirm their generalizability.

Adult identity mentoring: reducing sexual risk for African-American seventh grade students.

PURPOSE: This study was undertaken to determine whether the Adult Identity Mentoring (AIM) project successfully promotes abstinence, delays initiation of sex, and decreases intention to engage in sex. METHODS: Twenty middle school classes of African-American seventh graders were randomly assigned to receive either the AIM intervention or a standard health education control curriculum. The AIM is a 10-session curriculum based on the theory of possible selves. Class exercises encourage students to articulate a possible future self-identity and to develop self-promotion skills. Surveys about sexual activity were conducted before the intervention, 19 weeks after baseline, and again at 1 year after the intervention. RESULTS: Hierarchical logistic regression analyses showed significant effects for the intervention on sexual intentions, abstinence, and a trend toward fewer virgins initiating intercourse for the first time, 19 weeks after baseline. Specifically, students who received the intervention showed decreased intention to engage in sex and increased abstinence compared with students not receiving the intervention. Effects for 1-year follow-up, with smaller sample size, showed only that AIM male participants maintained the significant abstinence effect. CONCLUSIONS: A new intervention, AIM was evaluated among African-American seventh graders. This program, by focusing students on positive future selves, effectively modified sexual risk without directly providing instruction on sexually explicit topics.

Clustered Outbreak of Adverse Reactions to a Salsa Containing High Levels of Sulfites.

Six patients came to our attention in the Sacramento, CA area who developed cough or asthma after the ingestion of one brand (Brand 1) of refrigerated Mexican-style salsa, labelled "fresh". These patients had consumed other brands previously, not necessarily sulfited, but noted cough or asthma only in relation to consumption of this brand. Patients 1 and 2 developed reproducible severe cough and throat tightness after ingestion. Patients 3 and 4 developed status asthmaticus within minutes of ingesting salsa, requiring intubation and mechanical ventilation. Patients 5 and 6 experienced mild flares of their asthma after eating the salsa. The mean sulfite concentration of 6 containers of Brand 1 salsa was 1,803 ppm (range 1,630 to 1,920 ppm). Two other brands of salsa averaged 766 and 373 ppm sulfite, respectively. The manufacturer of Brand 1 salsa eliminated sulfites from their product after learning of the adverse reactions. These cases suggest that the amount of sulfite used was unnecessarily and dangerously high, and for one known sulfite-sensitive asthmatic, the use of the term "fresh" was misleading, resulting in near-fatal bronchospasm. Manufacturers should be careful to use only the minimum amount of sulfiting agent necessary.