Benefits of applying molecular barcoding systems are not uniform across different genomic applications.

BACKGROUND: Despite the wide variety of Next Generation Sequencing (NGS)-based methods, it remains challenging to detect mutations present at very low frequencies. This problem is particularly relevant in oncology, where the limiting amount of input material, and its low quality, often limit the performance of the assays. Unique Molecular Identifiers (UMIs) are a molecular barcoding system often coupled with computational methods of noise suppression to improve the reliability of detection of rare variants. Although widely adopted, UMI inclusion imposes additional technical complexity and sequencing cost. Currently, there are no guidelines on UMI usage nor a comprehensive evaluation of their advantage across different applications. METHODS: We used DNA sequencing data generated by molecular barcoding and hybridization-based enrichment, from various types and quantities of input material (fresh frozen, formaldehyde-treated and cell-free DNA), to evaluate the performance of variant calling in different clinically relevant contexts. RESULTS: Noise suppression achieved by read grouping based on fragment mapping positions ensures reliable variant calling for many experimental designs even without exogenous UMIs. Exogenous barcodes significantly improve performance only when mapping position collisions occur, which is common in cell-free DNA. CONCLUSIONS: We demonstrate that UMI usage is not universally beneficial across experimental designs and that it is worthwhile to critically consider the comparative advantage of UMI usage for a given NGS application prior to experimental design.

Mutational Hotspot in the SARS-CoV-2 Spike Protein N-Terminal Domain Conferring Immune Escape Potential.

Global efforts are being made to monitor the evolution of SARS-CoV-2, aiming for early identification of genotypes providing increased infectivity or virulence. However, viral lineage-focused tracking might fail in early detection of advantageous mutations emerging independently across phylogenies. Here, the emergence patterns of Spike mutations were investigated in sequences deposited in local and global databases to identify mutational hotspots across phylogenies and we evaluated their impact on SARS-CoV-2 evolution. We found a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. These mutations might have an impact on the evasion of neutralizing antibodies. Finally, we found that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path in which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape. We conclude that adaptive mutations, frequently present outside of the receptor-binding domain, can emerge in virtually any SARS-CoV-2 lineage and at any geographical location. Therefore, surveillance should not be restricted to monitoring defined lineages alone.

Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples.

OBJECTIVES: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping. METHODS: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination. RESULTS: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020. CONCLUSIONS: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.

Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor.

Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.

Opposing chromatin remodelers control transcription initiation frequency and start site selection.

Precise nucleosome organization at eukaryotic promoters is thought to be generated by multiple chromatin remodeler (CR) enzymes and to affect transcription initiation. Using an integrated analysis of chromatin remodeler binding and nucleosome occupancy following rapid remodeler depletion, we investigated the interplay between these enzymes and their impact on transcription in yeast. We show that many promoters are affected by multiple CRs that operate in concert or in opposition to position the key transcription start site (TSS)-associated +1 nucleosome. We also show that nucleosome movement after CR inactivation usually results from the activity of another CR and that in the absence of any remodeling activity, +1 nucleosomes largely maintain their positions. Finally, we present functional assays suggesting that +1 nucleosome positioning often reflects a trade-off between maximizing RNA polymerase recruitment and minimizing transcription initiation at incorrect sites. Our results provide a detailed picture of fundamental mechanisms linking promoter nucleosome architecture to transcription initiation.

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes.

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation.

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes.

The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.

Sequence-Directed Action of RSC Remodeler and General Regulatory Factors Modulates +1 Nucleosome Position to Facilitate Transcription.

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

Establishing nucleosome architecture and stability at promoters: Roles of pioneer transcription factors and the RSC chromatin remodeler.

Improvements in deep sequencing, together with methods to rapidly deplete essential transcription factors (TFs) and chromatin remodelers, have recently led to a more detailed picture of promoter nucleosome architecture in yeast and its relationship to transcriptional regulation. These studies revealed that ∼40% of all budding yeast protein-coding genes possess a unique promoter structure, where we propose that an unusually unstable nucleosome forms immediately upstream of the transcription start site (TSS). This "fragile" nucleosome (FN) promoter architecture relies on the combined action of the essential RSC (Remodels Structure of Chromatin) nucleosome remodeler and pioneer transcription factors (PTFs). FNs are associated with genes whose expression is high, coupled to cell growth, and characterized by low cell-to-cell variability (noise), suggesting that they may promote these features. Recent studies in metazoans suggest that the presence of dynamic nucleosomes upstream of the TSS at highly expressed genes may be conserved throughout evolution.

A Reply to "MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers," by Chereji et al.

In this issue of Molecular Cell, Chereji et al. (2017) present new data on MNase-sensitive particles previously identified upstream of transcription start sites at many promoters in budding yeast, and they argue, based upon negative histone-ChIP results, that they are non-nucleosomal signals generated by transcription factors (TFs). We show instead, based upon functional experiments where the relevant TFs are rapidly depleted, that this explanation does not hold, and we argue instead that histone ChIP and chemical cleavage assays have a limited capacity to capture these highly dynamic, MNase-sensitive "fragile" nucleosomes.

Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast.

Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.

Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.

Cleavage of the antitoxin of the parD toxin-antitoxin system is determined by the ClpAP protease and is modulated by the relative ratio of the toxin and the antitoxin.

Differential stability of toxins and antitoxins is the key for the conditional activation and function of Toxin-Antitoxin systems. Here we report the evaluation of the action of cell proteases Lon, ClpAP, ClpXP and ClpYQ on the Kis antitoxin and the Kid toxin of the parD TA system of plasmid R1. In vitro analysis shows that Kis antitoxin, but not the Kid toxin, is cleaved specifically by the ClpAP protease. The Kid toxin is not cleaved either by this protease or by any of the others cell proteases tested but in complex with the Kis antitoxin protects the cleavage of this protein in a way that is dependent on the toxin-antitoxin ratio. We further show that this protection is correlated with the inability of the ClpA chaperone to access the Kis antitoxin when in complex with Kid toxin. The stability of the antitoxin greatly increases in vivo in a clpP- background and plasmid maintenance mediated by the parD system, which is dependent on the differential decay of the antitoxin, is reduced to the levels observed in the absence of a functional toxin. The functional implications of these data are further discussed within the frame of the regulation of the parD system and of the available information on the nature of the toxin-antitoxin complexes formed at different toxin-antitoxin ratios.

Opposing effects of DNA on proteolysis of a replication initiator.

DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.

Replication and partitioning of the broad-host-range plasmid RK2.

The broad-host-range plasmid RK2 has been a model for studying DNA metabolism in bacteria for many years. It is used as a vector allowing genetic manipulations in numerous bacterial species. The RK2 genome encodes several genes providing the plasmid with diverse functions allowing for its stable maintenance in a variety of bacterial hosts. This review will focus on two processes indispensable for plasmid DNA maintenance. We will summarize recent understanding of the molecular mechanisms contributing to the RK2 DNA replication and partitioning.