Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation

Mouse cells package heterochromatin into compact foci. Erdel et al. show that these foci lack hallmarks of liquid droplets and rather resemble collapsed polymer globules. Their size, accessibility, and compaction are independent of HP1. They can adopt two distinct folding states that possibly represent the fundamental modes of chromatin compaction

Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes

TFIIH is an elongation factor of RNA polymerase I

TFIIH is a multisubunit factor essential for transcription initiation and promoter escape of RNA polymerase II and for the opening of damaged DNA double strands in nucleotide excision repair (NER). In this study, we have analyzed at which step of the transcription cycle TFIIH is essential for transcription by RNA polymerase I. We demonstrate that TFIIH associates with the rDNA promoter and gene-internal sequences and leaves the rDNA promoter in a complex with RNA polymerase I after start of transcription. Moreover, mutations in the TFIIH subunits XPB and XPD found in Cockayne syndrome impair the interaction of TFIIH with the rDNA, but do not influence initiation complex formation or promoter escape of RNA polymerase I, but preclude the productivity of the enzyme by reducing transcription elongation in vivo and in vitro. Our results implicate that reduced RNA polymerase I transcription elongation and ribosomal stress could be one factor contributing to the Cockayne syndrome phenotype

Transport emissions in Beijing: A scenario planning approach

This paper explores and analyses how to reduce smog-related air pollutants and carbon dioxide emissions generated by passenger transport systems in Beijing. In-depth surveys with experts and practitioners in China are used to examine the current business-as-usual projection for emissions in Beijing, the drivers and trends affecting current projections, and to develop alternative scenarios that might help reduce projected emissions significantly. These are based around different variants of population and migration growth and environmental stewardship. Current levels of smog caused by transport emissions are much higher in Beijing than internationally accepted safety standards, partly because of high levels of motorised traffic. Carbon dioxide emissions always tend to be overlooked because economic growth is prioritised. The sustainable model represents one of the best models for Beijing to follow; however, Beijing faces major challenges in becoming more environmentally sustainable over the next few years, mainly due to population growth and increased migration, even if there is powerful top-down government environmental stewardship. The aspiration to reduce smog-related air pollutants and carbon dioxide emissions in Beijing by implementing sustainable transport mitigation measures seems very ambitious; however, it is perhaps in this context that the real innovations in transport planning will emerge

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential "prion-like" domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59's localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of howCDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form

Adherence to Behavioural Interventions in Multiple Sclerosis: Follow-Up Meeting Report (AD@MS-2)

After an initial meeting in 2013 that reviewed adherence to disease modifying therapy, the AD@MS group conducted a follow-up meeting in 2014 that examined adherence to behavioural interventions in MS (e.g. physical activity, diet, psychosocial interventions). Very few studies have studied adherence to behavioural interventions in MS. Outcomes beyond six months are lacking, as well as implementation work in the community. Psychological interventions need to overcome stigma and other barriers to facilitate initiation and maintenance of behaviour change. A focus group concentrated on physical activity and exercise as one major behavioural intervention domain in MS. The discussion revealed that patients are confronted with multiple challenges when attempting to regularly engage in physical activity. Highlighted needs for future research included an improved understanding of patients’ and health experts’ knowledge and attitudes towards physical activity as well as a need for longitudinal research that investigates exercise persistence

Métamorphoses d’une utopie

Au moment où s’estompent au Canada la notion des « peuples fondateurs » et aux États-Unis celle de « melting pot » travaillées parfois jusqu’à l’érosion par les progrès civiques, il nous est paru urgent de penser les configurations identitaires à venir. Le multiculturalisme, l’interculture, le métissage, le « salad bowl », la transculture apparaissent alors comme autant de métamorphoses de l’utopie originelle : celle que les colons européens ont cherché à fonder en la déniant parfois aux peuples autochtones. Ainsi se pose à nouveau, au-delà des inévitables tentations communautaires, le rapport au politique, à la culture et enfin à l’État

Regulation of TCRbeta gene assembly by a promoter/enhancer holocomplex.

Antigen receptor gene assembly is governed by transcriptional promoters and enhancers that communicate over large distances and modulate chromatin accessibility to V(D)J recombinase. The precise role of these cis-acting elements in opening chromatin at recombinase targets and the mechanisms underlying their crosstalk remain unclear. We show that the TCRbeta enhancer (Ebeta) directs long-range chromatin opening over both DbetaJbeta clusters. Strikingly, chromatin associated with the Dbeta1 gene segment is refractory to Ebeta-mediated opening. Accessibility at Dbeta1 is accompanied by the formation of a stable holocomplex between a Dbeta-proximal promoter and Ebeta. These findings indicate a stepwise process for Dbeta --> Jbeta recombination that relies on distinct aspects of Ebeta activity: an intrinsic function that directs general chromatin opening and a cooperative function that facilitates the assembly of a promoter/enhancer holocomplex, unmasks the Dbeta1 gene segment, and triggers TCRbeta gene assembly