Biology subjects

McAnarney, E.

Publications and source records attributed to McAnarney, E..

3 recordsLinked to original sources

bioRxiv2022

Nucleosome conformation dictates the histone code

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized codes that are read by specialized regions (reader domains) in chromatin associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP-histone PTM] specificity, and thus decipher the histone code / guide epigenetic therapies. However, this has largely been done using a reductive approach of isolated reader domains and histone peptides, with the assumption that PTM readout is unaffected by any higher order factors. Here we show that CAP-histone PTM interaction is in fact dependent on nucleosome context. Our results indicate this is due to histone tail accessibility and the associated impact on binding potential of reader domains. We further demonstrate that the in vitro specificity of a tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. This necessitates we refine the histone code concept and interrogate it at the nucleosome level.

genomics↗

bioRxiv2020

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

biochemistry↗

bioRxiv2020

Topoisomerase III-beta is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-{beta} (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

microbiology↗