The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms.

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription.

Drawing from and Expanding their Toolboxes: Preschool Teachers' Traditional Strategies, Unconventional Opportunities, and Novel Challenges in Scaffolding Young Children's Social and Emotional Learning During Remote Instruction Amidst COVID-19.

Building on aspects of Vygotsky's sociocultural theory centering around social interaction and adult scaffolding as essential to children's learning, this study investigated the most prominently used strategies by eight teachers to scaffold social and emotional learning (SEL) in preschool children (ages 3-4) in the context of remote instruction during the 2021-2022 school year amidst COVID-19. These teachers (seven females and one male) came from two urban preschools funded by their local Board of Education in the state of New Jersey in the United States. These teachers (ages 28-44 years, M = 32 years) varied in teaching experience from five to 29 years (M = 13 years). Each teacher was interviewed for an average of 40 min virtually via Zoom. The interviews were digitally recorded and then transcribed for analysis. A thematic analysis of the data revealed that the three most salient strategies the teachers implemented to virtually scaffold the children's SEL were: (1) involving book reading and discussion, (2) utilizing visuals, and (3) engaging in targeted conversations. In addition to adapting these three traditional strategies applied during in-person instruction to remote instruction, the teachers creatively and appropriately leveraged online resources to further scaffold and enhance children's SEL in the unconventional virtual environment, thereby expanding their toolboxes. Despite their intentional efforts, these teachers found that there were unconventional opportunities and novel challenges in scaffolding children's SEL during remote instruction not traditionally found during in-person instruction. Collectively, the findings of this study suggest that in-person instruction, due to its social nature, is still the most optimal condition for promoting children's SEL.

Decoding histone ubiquitylation.

Histone ubiquitylation is a critical part of both active and repressed transcriptional states, and lies at the heart of DNA damage repair signaling. The histone residues targeted for ubiquitylation are often highly conserved through evolution, and extensive functional studies of the enzymes that catalyze the ubiquitylation and de-ubiquitylation of histones have revealed key roles linked to cell growth and division, development, and disease in model systems ranging from yeast to human cells. Nonetheless, the downstream consequences of these modifications have only recently begun to be appreciated on a molecular level. Here we review the structure and function of proteins that act as effectors or "readers" of histone ubiquitylation. We highlight lessons learned about how ubiquitin recognition lends specificity and function to intermolecular interactions in the context of transcription and DNA repair, as well as what this might mean for how we think about histone modifications more broadly.

Communicating Digitally: Building Preschool Teacher-Parent Partnerships Via Digital Technologies During COVID-19.

During COVID-19, many schools in the United States restrict parent visits and parent-teacher face-to-face meetings. Consequently, teachers and parents rely on digital technologies to communicate and build partnerships. Yet, little is known about their perceived experiences with digital communication. To contribute knowledge to this area, this study investigated the perceptions of the classroom teacher and parents of preschool children concerning their experiences of communicating with each other via digital technologies during COVID-19. The participants consisted of one teacher and three mothers of 3-year-olds in the same classroom of a private childcare center serving preschool children from mostly middle-class backgrounds in a northeastern state of the United States. The teacher and parents were interviewed individually and virtually via Zoom for 30-60 min (M = 45 min). A thematic analysis uncovered four salient themes: (1) modes of digital communication between the teacher and parents, (2) the nature of digital communication, (3) limitations of digital communication, and (4) digital communication via ClassDojo. The ClassDojo theme further revealed three subthemes: (1) ClassDojo for promoting proactive parent involvement, (2) ClassDojo for building teacher-parent partnerships, and (3) the use of limited functions of ClassDojo. The data were triangulated by analyzing teacher-parent communication artifacts on ClassDojo, which confirmed the findings related to the use of this digital platform.

Thriving Beyond Resilience Despite Stress: A Psychometric Evaluation of the Newly Developed Teacher Stress Scale and Teacher Thriving Scale.

Building on theoretical and empirical insights and applying the thriving theory as the conceptual framework, the authors developed two new teacher-specific scales, namely the Teacher Stress Scale (TSS) and the Teacher Thriving Scale (TTS). The goal of this investigation was to evaluate the psychometric properties of these two scales. Data were collected through an online questionnaire administered to a national sample of 122 participating early childhood teachers (ages 22-72 years, M = 41.01) teaching in preschool through third grade in 26 states of the United States during the 2020-2021 school year amidst COVID-19. This study revealed some important psychometric results. First, with respect to their internal structures, both the TSS and the TTS appeared to be best represented as bifactorial and trifactorial, respectively. Specifically, the TSS comprised two constructs: (1) Inadequate School-based Support, and (2) Teaching-related Demands; and the TTS encompassed three constructs: (1) Adaptability and Flexibility, (2) Personal Strengths and Professional Growth, and (3) Positive Mindset. Second, the negative correlation between the TSS and the TTS provided discriminant evidence for each other's construct validity, while the positive correlations between the TTS and six conceptually cognate constructs (Stress Resilience, Resilience Coping, Coping Efficacy, Teaching Satisfaction, Emotional Support, and Gratitude) demonstrated convergent evidence for construct validity for the TTS. Third, both the overall TSS and the overall TTS as well as their subscales exhibited good internal consistency reliability. Fourth, both the overall TSS and the overall TTS also demonstrated test-retest reliability.

A role for BET proteins in regulating basal, dopamine-induced and cAMP/PKA-dependent transcription in rat striatal neurons.

The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a Gαs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

Learning gain rather than learning loss during COVID-19: A proposal for reframing the narrative.

"Learning loss" has become the new buzzword in education during the COVID-19 era. Learning loss may be real in certain academic subjects (e.g. mathematics and reading) for certain students, as indicated by standardized test scores. However, it only tells a partial story. The other part of the story actually indicates different kinds of learning gain that might have occurred for children experiencing non-conventional learning opportunities during the COVID-19 pandemic. Thus, the authors caution against subscribing to a learning-loss narrative, a deficits-based perspective, which can lead one to lose sight of children's potential learning gains that are not necessarily assessed or recognized. Against this backdrop, the authors offer four recommendations: (1) reframing the concept of "learning loss" to "learning gain"; (2) applying a strengths-based model rather than a deficits-based model for understanding student learning; (3) investing in the development of the whole child; and (4) ensuring that we focus on young children's socio-emotional well-being (e.g. relationship-building) and not solely on the cognitive domains.

Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms.

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

The Supramap project: linking pathogen genomes with geography to fight emergent infectious diseases.

Novel pathogens have the potential to become critical issues of national security, public health and economic welfare. As demonstrated by the response to Severe Acute Respiratory Syndrome (SARS) and influenza, genomic sequencing has become an important method for diagnosing agents of infectious disease. Despite the value of genomic sequences in characterizing novel pathogens, raw data on their own do not provide the information needed by public health officials and researchers. One must integrate knowledge of the genomes of pathogens with host biology and geography to understand the etiology of epidemics. To these ends, we have created an application called Supramap (http://supramap.osu.edu) to put information on the spread of pathogens and key mutations across time, space and various hosts into a geographic information system (GIS). To build this application, we created a web service for integrated sequence alignment and phylogenetic analysis as well as methods to describe the tree, mutations, and host shifts in Keyhole Markup Language (KML). We apply the application to 239 sequences of the polymerase basic 2 (PB2) gene of recent isolates of avian influenza (H5N1). We map a mutation, glutamic acid to lysine at position 627 in the PB2 protein (E627K), in H5N1 influenza that allows for increased replication of the virus in mammals. We use a statistical test to support the hypothesis of a correlation of E627K mutations with avian-mammalian host shifts but reject the hypothesis that lineages with E627K are moving westward. Data, instructions for use, and visualizations are included as supplemental materials at: http://supramap.osu.edu/sm/supramap/publications. © The Willi Hennig Society 2010.

Medicare Part D prescription drug benefits and administrative burden in the care of dually eligible psychiatric patients.

OBJECTIVE: With implementation of Medicare Part D, concerns were raised that patients with severe mental illness who were dually eligible for both Medicaid and Medicare benefits would be at clinical risk. In addition to concerns about medication access and continuity, there were concerns about administrative burden for physicians and their staffs. This study aimed to quantify the amount of administrative burden for psychiatrists and their staff related to Medicare Part D prescription drug plan administration in a national sample of dually eligible psychiatric patients and to identify factors associated with increased burden. METHODS: A total of 5,833 psychiatrists were randomly selected from the American Medical Association's Physicians Masterfile. Responses were obtained from 64% (N=3,247) with a mailed survey using practice-based survey research methods during the first four months of Medicare Part D implementation (January to April 2006); 1,183 psychiatrists met eligibility requirements. RESULTS: Psychiatrists and their staff spent 45 minutes in administrative tasks for every one hour of direct patient care for dually eligible patients. Drug plan features, including prior authorization and preferred drug formularies, and medication access problems were associated with increased administrative time. CONCLUSIONS: Results of this study indicate several drug plan features and medication access problems related to Part D implementation were associated with significant increases in administrative burden for psychiatrists and their staff, which may result in less time for direct patient care. Given the vulnerability of this high-risk population, this increased administrative burden may pose a significant risk to the overall quality of care for psychiatric patients.