RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

Defining the importance of landscape metrics for large branchiopod biodiversity and conservation: the case of the Iberian Peninsula and Balearic Islands

The deficiency in the distributional data of invertebrate taxa is one of the major impediments acting on the bias towards the low awareness of its conservation status. The present study sets a basic framework to understand the large branchiopods distribution in the Iberian Peninsula and Balearic Islands. Since the extensive surveys performed in the late 1980s, no more studies existed updating the information for the whole studied area. The present study fills the gap, gathering together all available information on large branchiopods distribution since 1995, and analysing the effect of human population density and several landscape characteristics on their distribution, taking into consideration different spatial scales (100 m, 1 km and 10 km). In overall, 28 large branchiopod taxa (17 anostracans, 7 notostracans and 4 spinicaudatans) are known to occur in the area. Approximately 30% of the sites hosted multiple species, with a maximum of 6 species. Significant positive co-occurring species pairs were found clustered together, forming 4 different associations of large branchiopod species. In general, species clustered in the same group showed similar responses to analysed landscape characteristics, usually showing a better fit at higher spatial scales.Brazilian Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq [401045/2014-5]Spanish Ministry of Education, Culture and Sport [FPU014/06783]info:eu-repo/semantics/publishedVersio

The Atypical Calpains: Evolutionary Analyses and Roles in Caenorhabditis elegans Cellular Degeneration

The calpains are physiologically important Ca2+-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca2+]i. We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca2+ dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover

Gene regulatory network inference from systems genetics data using tree-based methods

peer reviewedOne of the pressing open problems of computational systems biology is the elucidation of the topology of gene regulatory networks (GRNs). In an attempt to solve this problem, the idea of systems genetics is to exploit the natural variations that exist between the DNA sequences of related individuals and that can represent the randomized and multifactorial perturbations necessary to recover GRNs. In this chapter, we present new methods, called GENIE3-SG-joint and GENIE3- SG-sep, for the inference of GRNs from systems genetics data. Experiments on the artificial data of the StatSeq benchmark and of the DREAM5 Systems Genetics challenge show that exploiting jointly expression and genetic data is very helpful for recovering GRNs, and one of our methods outperforms by a large extent the official best performing method of the DREAM5 challenge

Benchmarking atlas-level data integration in single-cell genomics

AbstractCell atlases often include samples that span locations, labs, and conditions, leading to complex, nested batch effects in data. Thus, joint analysis of atlas datasets requires reliable data integration.Choosing a data integration method is a challenge due to the difficulty of defining integration success. Here, we benchmark 38 method and preprocessing combinations on 77 batches of gene expression, chromatin accessibility, and simulation data from 23 publications, altogether representing &gt;1.2 million cells distributed in nine atlas-level integration tasks. Our integration tasks span several common sources of variation such as individuals, species, and experimental labs. We evaluate methods according to scalability, usability, and their ability to remove batch effects while retaining biological variation.Using 14 evaluation metrics, we find that highly variable gene selection improves the performance of data integration methods, whereas scaling pushes methods to prioritize batch removal over conservation of biological variation. Overall, BBKNN, Scanorama, and scVI perform well, particularly on complex integration tasks; Seurat v3 performs well on simpler tasks with distinct biological signals; and methods that prioritize batch removal perform best for ATAC-seq data integration. Our freely available reproducible python module can be used to identify optimal data integration methods for new data, benchmark new methods, and improve method development.</jats:p