In vivo analysis reveals that ATP-hydrolysis couples remodeling to SWI/SNF release from chromatin

ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.

In vivo Analysis Reveals that ATP-hydrolysis Couples Remodeling to SWI/SNF Release from Chromatin

ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.

Potent CRISPR-Cas9 inhibitors fromStaphylococcusgenomes

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibitingStaphylococcus aureusCas9 (SauCas9), an alternative to the most commonly used genome editing proteinStreptococcus pyogenesCas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13–AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13–AcrIIA15 as unique bifunctional inhibitors of SauCas9.

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three new potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14 and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for anti-CRISPR function, and have divergent C-termini that are required in each case for selective inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust and specific inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-15 as unique bifunctional inhibitors of SauCas9.

Phylogenomics provides new insights into gains and losses of selenoproteins among Archaeplastida

Selenoproteins that contain selenocysteine (Sec) are found in all kingdoms of life. Although they constitute a small proportion of the proteome, selenoproteins play essential roles in many organisms. In photosynthetic eukaryotes, selenoproteins have been found in algae but are missing in land plants (embryophytes). In this study, we explored the evolutionary dynamics of Sec incorporation by conveying a genomic search for the Sec machinery and selenoproteins across Archaeplastida. We identified a complete Sec machinery and variable sizes of selenoproteomes in the main algal lineages. However, the entire Sec machinery was missing in the BV clade (Bangiophyceae-Florideophyceae) of Rhodoplantae (red algae) and only partial machinery was found in three species of Archaeplastida, indicating parallel loss of Sec incorporation in different groups of algae. Further analysis of genome and transcriptome data suggests that all major lineages of streptophyte algae display a complete Sec machinery, although the number of selenoproteins is low in this group, especially in subaerial taxa. We conclude that selenoproteins tend to be lost in Archaeplastida upon adaptation to a subaerial or acidic environment. The high number of redox-active selenoproteins found in some bloom-forming marine microalgae may be related to defense against viral infections. Some of the selenoproteins in these organisms may have been gained by horizontal gene transfer from bacteria.

Phylogenomics Provides New Insights into Gains and Losses of Selenoproteins among Archaeplastida

Selenoproteins that contain selenocysteine (Sec) are found in all kingdoms of life. Although they constitute a small proportion of the proteome, selenoproteins play essential roles in many organisms. In photosynthetic eukaryotes, selenoproteins have been found in algae but are missing in land plants (embryophytes). In this study, we explored the evolutionary dynamics of Sec incorporation by conveying a genomic search for the Sec machinery and selenoproteins across Archaeplastida. We identified a complete Sec machinery and variable sizes of selenoproteomes in the main algal lineages. However, the entire Sec machinery was missing in the Bangiophyceae-Florideophyceae clade (BV) of Rhodoplantae (red algae) and only partial machinery was found in three species of Archaeplastida, indicating parallel loss of Sec incorporation in different groups of algae. Further analysis of genome and transcriptome data suggests that all major lineages of streptophyte algae display a complete Sec machinery, although the number of selenoproteins is low in this group, especially in subaerial taxa. We conclude that selenoproteins tend to be lost in Archaeplastida upon adaptation to a subaerial or acidic environment. The high number of redox-active selenoproteins found in some bloom-forming marine microalgae may be related to defense against viral infections. Some of the selenoproteins in these organisms may have been gained by horizontal gene transfer from bacteria.

synder: inferring genomic orthologs from synteny maps

Ortholog inference is a key step in understanding the evolution and function of a gene or other genomic feature. Yet often no similar sequence can be identified, or the true ortholog is hidden among false positives. A solution is to consider the sequence’s genomic context. We present the generic program,synder, for tracing features of interest between genomes based on a synteny map. This approach narrows genomic search-space independently of the sequence of the feature of interest. We illustrate the utility ofsynderby finding orthologs for theArabidopsis thaliana13-member gene family of Nuclear Factor YC transcription factor across the Brassicaceae clade.