The Mi-2 Homolog Mit1 Actively Positions Nucleosomes Within Heterochromatin to Suppress Transcription

Eukaryotic DNA is packaged into the nucleus in complex with proteins that regulate access and utilization of the genetic material. These DNA and protein complexes form a dynamic structure known as chromatin. Nucleosomes are the repeating unit of chromatin, and consist of DNA wrapped around an octamer of histone proteins. Nucleosomes can then be modified and spatially arranged to facilitate processes such as transcription, DNA replication, and repair. A special transcriptionally repressive chromatin structure assembles onto gene-poor, repetitive regions of the genome known as constitutive heterochromatin. Mit1 is the putative chromatin remodeling subunit of the fission yeast Snf2/HDAC repressor complex (SHREC) and is known to repress transcription at regions of heterochromatin. However, how Mit1 modifies chromatin to silence transcription is largely unknown. Here we report that Mit1 mobilizes histone octamers in vitro Document Type: Doctoral Dissertation Name: Kevin M. Creamer Email Address: [email protected] Title: The Mi-2 Homolog Mit1 Actively Positions Nucleosomes within Heterochromatin to Suppress Transcription Degree: Doctor of Philosophy Program (Major): Biomedical Sciences Concentration (Track): Cell Biology and Biochemistry Research Advisor: Janet F. Partridge, Ph.D. Advisor\u27s Email: [email protected] Committee Members: Mark Bix, Ph.D. Paul K. Brindle, Ph.D. Linda M. Hendershot, Ph.D. Ronald N. Laribee, Ph.D. Keywords: chromatin remodeling, fission yeast, heterochromatin, Mit1, SHREC Availability: Embargoed for 12 months Graduation Date: May 2014 and requires ATP hydrolysis and conserved chromatin tethering domains including a previously unrecognized chromodomain to remodel nucleosomes and silence transcription. Loss of Mit1 remodeling activity results in nucleosome depletion at specific DNA sequences that display low intrinsic affinity for the histone octamer, but its contribution to antagonizing RNA Polymerase II access and transcription is not restricted to these sites. Genetic epistasis analyses demonstrate that SHREC subunits and the transcription coupled Set2 histone methyltransferase, which is involved in suppression of cryptic transcription at actively transcribed regions, cooperate to silence heterochromatic transcripts. In addition, we demonstrate that Mit1’s remodeling activity contributes to SHREC function independently of Clr3’s histone deacetylase activity on Lys14 of histone H3. We propose that chromatin remodeling by Mit1 cooperates with the Clr3 and other chromatin modifiers to stabilize heterochromatin structure and to prevent access to the transcriptional machinery

XIST RNA: a window into the broader role of RNA in nuclear chromosome architecture

XIST RNA triggers the transformation of an active X chromosome into a condensed, inactive Barr body and therefore provides a unique window into transitions of higher-order chromosome architecture. Despite recent progress, how XIST RNA localizes and interacts with the X chromosome remains poorly understood. Genetic engineering of XIST into a trisomic autosome demonstrates remarkable capacity of XIST RNA to localize and comprehensively silence that autosome. Thus, XIST does not require X chromosome-specific sequences but operates on mechanisms available genome-wide. Prior results suggested XIST localization is controlled by attachment to the insoluble nuclear scaffold. Our recent work affirms that scaffold attachment factor A (SAF-A) is involved in anchoring XIST, but argues against the view that SAF-A provides a unimolecular bridge between RNA and the chromosome. Rather, we suggest that a complex meshwork of architectural proteins interact with XIST RNA. Parallel work studying the territory of actively transcribed chromosomes suggests that repeat-rich RNA \u27coats\u27 euchromatin and may impact chromosome architecture in a manner opposite of XIST A model is discussed whereby RNA may not just recruit histone modifications, but more directly impact higher-order chromatin condensation via interaction with architectural proteins of the nucleus.This article is part of the themed issue \u27X-chromosome inactivation: a tribute to Mary Lyon\u27

Correcting motion induced fluorescence artifacts in two-channel neural imaging

Imaging neural activity in a behaving animal presents unique challenges in part because motion from an animal's movement creates artifacts in fluorescence intensity time-series that are difficult to distinguish from neural signals of interest. One approach to mitigating these artifacts is to image two channels; one that captures an activity-dependent fluorophore, such as GCaMP, and another that captures an activity-independent fluorophore such as RFP. Because the activity-independent channel contains the same motion artifacts as the activity-dependent channel, but no neural signals, the two together can be used to remove the artifacts. Existing approaches for this correction, such as taking the ratio of the two channels, do not account for channel independent noise in the measured fluorescence. Moreover, no systematic comparison has been made of existing approaches that use two-channel signals. Here, we present Two-channel Motion Artifact Correction (TMAC), a method which seeks to remove artifacts by specifying a generative model of the fluorescence of the two channels as a function of motion artifact, neural activity, and noise. We further present a novel method for evaluating ground-truth performance of motion correction algorithms by comparing the decodability of behavior from two types of neural recordings; a recording that had both an activity-dependent fluorophore (GCaMP and RFP) and a recording where both fluorophores were activity-independent (GFP and RFP). A successful motion-correction method should decode behavior from the first type of recording, but not the second. We use this metric to systematically compare five methods for removing motion artifacts from fluorescent time traces. We decode locomotion from a GCaMP expressing animal 15x more accurately on average than from control when using TMAC inferred activity and outperform all other methods of motion correction tested.Comment: 11 pages, 3 figure

Collaborative Assessment and Survey Administration: A MISO Survey Case Study

The Managing Information Services Outcomes (MISO) Survey was originally developed in 2005 at Bryn Mawr College in Pennsylvania, USA by staff from a consortium of higher education institutions to assess library and technology services using a single instrument. Since then, the survey has grown and changed under the collaborative management of an all-volunteer team of library and IT professionals from various participating institutions throughout the United States. The survey has been implemented at 171 institutions. This chapter reviews the guiding principles, ongoing partnerships, and the value of working across departments, across campuses, and across cohorts of participating institutions to leverage local expertise, reduce costs, and create a culture of collaboration and assessment. In an environment where libraries are increasingly dependent on information technology, assessing library services in the context of IT brings layered intelligence to data-informed decision making. -- Chapter 9, p. 17

SAF-A mutants disrupt chromatin structure through dominant negative effects on RNAs associated with chromatin

Here we provide a brief review of relevant background before presenting results of our investigation into the interplay between scaffold attachment factor A (SAF-A), chromatin-associated RNAs, and DNA condensation. SAF-A, also termed heterogenous nuclear protein U (hnRNP U), is a ubiquitous nuclear scaffold protein that was implicated in XIST RNA localization to the inactive X-chromosome (Xi) but also reported to maintain open DNA packaging in euchromatin. Here we use several means to perturb SAF-A and examine potential impacts on the broad association of RNAs on euchromatin, and on chromatin compaction. SAF-A has an N-terminal DNA binding domain and C-terminal RNA binding domain, and a prominent model has been that the protein provides a single-molecule bridge between XIST RNA and chromatin. Here analysis of the impact of SAF-A on broad RNA-chromatin interactions indicate greater biological complexity. We focus on SAF-A\u27s role with repeat-rich C0T-1 hnRNA (repeat-rich heterogeneous nuclear RNA), shown recently to comprise mostly intronic sequences of pre-mRNAs and diverse long non-coding RNAs (lncRNAs). Our results show that SAF-A mutants cause dramatic changes to cytological chromatin condensation through dominant negative effects on C0T-1 RNA\u27s association with euchromatin, and likely other nuclear scaffold factors. In contrast, depletion of SAF-A by RNA interference (RNAi) had no discernible impact on C0T-1 RNA, nor did it cause similarly marked chromatin changes as did three different SAF-A mutations. Overall results support the concept that repeat-rich, chromatin-associated RNAs interact with multiple RNA binding proteins (RBPs) in a complex dynamic meshwork that is integral to larger-scale chromatin architecture and collectively influences cytological-scale DNA condensation

Building an eScience Thesaurus for Librarians: A Collaboration Between the National Network of Libraries of Medicine, New England Region and an Associate Fellow at the National Library of Medicine

Objective: In response to the growing interest and adoption of eScience roles by librarians, those from the National Network of Libraries of Medicine, New England Region (NN/LM NER) and an Associate Fellow from the National Library of Medicine collaborated to build an eScience Thesaurus. The Thesaurus will introduce librarians to terminology and concepts in eScience, point to relevant literature and resources on data and digital research topics, and provide links to interviews with librarians and experts working in eScience-related roles. The eScience Thesaurus is a starting place for librarians to find the vocabulary to research the background, resources, and tools necessary for developing their capacity to provide eScience-related services. Methods: The Associate Fellow completed a review of eScience-related literature to identify the seminal publications for the originations of these terms and concepts as they apply to libraries. Next, the Associate Fellow worked with the NN/LM NER to compile an environmental scan of resources that would be useful and applicable for librarians, and created a scope document and record structure. The team interviewed prominent librarians working in eScience roles and experts that have created digital tools and services used by the library community. Finally, the team sent the Thesaurus records out to five members of the advisory and editorial review boards from the eScience Portal for New England Librarians for evaluation. Results: The eScience Thesaurus is now hosted on the eScience Portal for New England Librarians’ website. It provides a comprehensive list of more than 50 different terminologies and concepts, with links to seminal and relevant literature, resources, grants, and interviews on a variety of eScience-related topics. Conclusion: The eScience Thesaurus is an evolving resource; as the field expands and more eScience-related terms are adopted by the library and information science community, the Portal will enable its users to electronically submit new vocabulary and records to the Thesuarus, thus preserving it as a go-to eScience resource for librarians

ERS International Congress 2023: highlights from the Thoracic Oncology Assembly

Lung cancer is the leading cause of cancer mortality in the world. It greatly affects the patients' quality of life, and is thus a challenge for the daily practice in respiratory medicine. Advances in the genetic knowledge of thoracic tumours' mutational landscape, and the development of targeted therapies and immune checkpoint inhibitors, have led to a paradigm shift in the treatment of lung cancer and pleural mesothelioma. During the 2023 European Respiratory Society Congress in Milan, Italy, experts from all over the world presented their high-quality research and reviewed best clinical practices. Lung cancer screening, management of early stages of lung cancer, application of artificial intelligence and biomarkers were discussed and they will be summarised here

Clinical effectiveness of rapid tests for methicillin resistant Staphylococcus aureus (MRSA) in hospitalized patients: a systematic review

<p>Abstract</p> <p>Background</p> <p>Methicillin resistant <it>Staphylococcus aureus </it>(MRSA) are often resistant to multiple classes of antibiotics. The research objectives of this systematic review were to evaluate the clinical effectiveness of polymerase chain reaction (PCR) versus chromogenic agar for MRSA screening, and PCR versus no screening for several clinical outcomes, including MRSA colonization and infection rates.</p> <p>Methods</p> <p>An electronic literature search was conducted on studies evaluating polymerase chain reaction techniques and methicillin (also spelled meticillin) resistant <it>Staphylococcus aureus </it>that were published from 1993 onwards using Medline, Medline In-Process & Other Non-Indexed Citations, BIOSIS Previews, and EMBASE. Due to the presence of heterogeneity in the selected studies, the clinical findings of individual studies were described.</p> <p>Results</p> <p>Nine studies that compared screening for MRSA using PCR versus screening using chromogenic agar in a hospital setting, and two studies that compared screening using PCR with no or targeted screening were identified. Some studies found lower MRSA colonization and acquisition, infection, and transmission rates in screening with PCR versus screening with chromogenic agar, and the turnaround time for screening test results was lower for PCR. One study reported a lower number of unnecessary isolation days with screening using PCR versus screening with chromogenic agar, but the proportion of patients isolated was similar between both groups. The turnaround time for test results and number of isolation days were lower for PCR versus chromogenic agar for MRSA screening.</p> <p>Conclusions</p> <p>The use of PCR for MRSA screening demonstrated a lower turnaround time and number of isolation days compared with chromogenic agar. Given the mixed quality and number of studies (11 studies), gaps remain in the published literature and the evidence remains insufficient. In addition to screening, factors such as the number of contacts between healthcare workers and patients, number of patients attended by one healthcare worker per day, probability of colonization among healthcare workers, and MRSA status of hospital shared equipment and hospital environment must be considered to control the transmission of MRSA in a hospital setting.</p

Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation

Phosphorylation of the kinetochore component Nsk1 by Cdk1 antagonizes its localization to and function at the kinetochore and spindle during early mitosis

Continuous Requirement for the Clr4 Complex But Not RNAi for Centromeric Heterochromatin Assembly in Fission Yeast Harboring a Disrupted RITS Complex

Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent “priRNAs.” The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi–independent factors in the assembly of heterochromatin