Dissecting the cis-regulatory syntax of transcription initiation with deep learning.

Despite extensive characterization of mammalian Pol II transcription, the DNA sequence determinants of transcription initiation at a third of human promoters and most enhancers remain poorly understood. Hence, we trained and interpreted a neural network called ProCapNet that accurately models base-resolution initiation profiles from PRO-cap experiments using local DNA sequence. ProCapNet learns sequence motifs with distinct effects on initiation rates and TSS positioning and uncovers context-specific cryptic initiator elements intertwined within other TF motifs. ProCapNet annotates predictive motifs in nearly all actively transcribed regulatory elements across multiple cell-lines, revealing a shared cis-regulatory logic across promoters and enhancers mediated by a highly epistatic sequence syntax of cooperative and competitive motif interactions. ProCapNet models of RAMPAGE profiles measuring steady-state RNA abundance at TSSs distill initiation signals on par with models trained directly on PRO-cap profiles. ProCapNet learns a largely cell-type-agnostic cis-regulatory code of initiation complementing sequence drivers of cell-type-specific chromatin state critical for accurate prediction of cell-type-specific transcription initiation.

An updated compendium and reevaluation of the evidence for nuclear transcription factor occupancy over the mitochondrial genome.

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

The killifish germline regulates longevity and somatic repair in a sex-specific manner.

Classical evolutionary theories propose tradeoffs among reproduction, damage repair and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. In this study, we used the turquoise killifish (Nothobranchius furzeri) to genetically arrest germline development at discrete stages and examine how different modes of infertility impact life history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. We show here that germline depletion-but not arresting germline differentiation-enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted Caenorhabditis elegans. Our results, therefore, demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs.

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.

BACKGROUND: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. RESULTS: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays. CONCLUSIONS: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.

Protocol for mapping the three-dimensional organization of dinoflagellate genomes.

Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al.1.

Single-molecule chromatin configurations link transcription factor binding to expression in human cells.

The binding of multiple transcription factors (TFs) to genomic enhancers activates gene expression in mammalian cells. However, the molecular details that link enhancer sequence to TF binding, promoter state, and gene expression levels remain opaque. We applied single-molecule footprinting (SMF) to measure the simultaneous occupancy of TFs, nucleosomes, and components of the transcription machinery on engineered enhancer/promoter constructs with variable numbers of TF binding sites for both a synthetic and an endogenous TF. We find that activation domains enhance a TF's capacity to compete with nucleosomes for binding to DNA in a BAF-dependent manner, TF binding on nucleosome-free DNA is consistent with independent binding between TFs, and average TF occupancy linearly contributes to promoter activation rates. We also decompose TF strength into separable binding and activation terms, which can be tuned and perturbed independently. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the binding microstates observed at the enhancer and subsequent time-dependent gene expression. This work provides a template for quantitative dissection of distinct contributors to gene activation, including the activity of chromatin remodelers, TF activation domains, chromatin acetylation, TF concentration, TF binding affinity, and TF binding site configuration.

The killifish germline regulates longevity and somatic repair in a sex-specific manner.

Classical evolutionary theories propose tradeoffs between reproduction, damage repair, and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. Here, we use the turquoise killifish ( N. furzeri ) to genetically arrest germline development at discrete stages, and examine how different modes of infertility impact life-history. We first construct a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. Next, we show that germline depletion - but not arresting germline differentiation - enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted C. elegans . Our results therefore demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs.

The landscape of the histone-organized chromatin of Bdellovibrionota bacteria.

Histone proteins have traditionally been thought to be restricted to eukaryotes and most archaea, with eukaryotic nucleosomal histones deriving from their archaeal ancestors. In contrast, bacteria lack histones as a rule. However, histone proteins have recently been identified in a few bacterial clades, most notably the phylum Bdellovibrionota, and these histones have been proposed to exhibit a range of divergent features compared to histones in archaea and eukaryotes. However, no functional genomic studies of the properties of Bdellovibrionota chromatin have been carried out. In this work, we map the landscape of chromatin accessibility, active transcription and three-dimensional genome organization in a member of Bdellovibrionota (a Bacteriovorax strain). We find that, similar to what is observed in some archaea and in eukaryotes with compact genomes such as yeast, Bacteriovorax chromatin is characterized by preferential accessibility around promoter regions. Similar to eukaryotes, chromatin accessibility in Bacteriovorax positively correlates with gene expression. Mapping active transcription through single-strand DNA (ssDNA) profiling revealed that unlike in yeast, but similar to the state of mammalian and fly promoters, Bacteriovorax promoters exhibit very strong polymerase pausing. Finally, similar to that of other bacteria without histones, the Bacteriovorax genome exists in a three-dimensional (3D) configuration organized by the parABS system along the axis defined by replication origin and termination regions. These results provide a foundation for understanding the chromatin biology of the unique Bdellovibrionota bacteria and the functional diversity in chromatin organization across the tree of life.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size.

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.

The chromatin landscape of the euryarchaeon Haloferax volcanii.

BACKGROUND: Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools. RESULTS: Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures. CONCLUSIONS: Our results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin.

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome.

In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties were originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.

CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

Genome-Wide Mapping of Active Regulatory Elements Using ATAC-seq.

Active cis-regulatory elements (cREs) in eukaryotes are characterized by nucleosomal depletion and, accordingly, higher accessibility. This property has turned out to be immensely useful for identifying cREs genome-wide and tracking their dynamics across different cellular states and is the basis of numerous methods taking advantage of the preferential enzymatic cleavage/labeling of accessible DNA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as the most versatile and widely adaptable method and has been widely adopted as the standard tool for mapping open chromatin regions. Here, we discuss the current optimal practices and important considerations for carrying out ATAC-seq experiments, primarily in the context of mammalian systems.

Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF.

The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.

Simultaneous Single-Cell Profiling of the Transcriptome and Accessible Chromatin Using SHARE-seq.

The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results.

The Impact and Progression of the COVID-19 Pandemic in Bulgaria in Its First Two Years.

After initially having low levels of SARS-CoV-2 infections for much of the year, Bulgaria experienced a major epidemic surge at the end of 2020, which caused the highest recorded excess mortality in Europe, among the highest in the word (Excess Mortality Rate, or EMR ∼0.25%). Two more major waves followed in 2021, followed by another one in early 2022. In this study, we analyze the temporal and spatial patterns of excess mortality at the national and local levels and across different demographic groups in Bulgaria and compare those to the European levels. Bulgaria has continued to exhibit the previous pattern of extremely high excess mortality, as measured both by crude mortality metrics (an EMR of ∼1.05%, up to the end of March 2022) and by standardized ones-Potential Years of Life Lost (PYLL) and Aged-Standardized Years of life lost Rate (ASYR). Unlike Western Europe, the bulk of excess mortality in Bulgaria, as well as in several other countries in Eastern Europe, occurred in the second year of the pandemic, likely related to the differences in the levels of vaccination coverage between these regions. We also observe even more extreme levels of excess mortality at the regional level and in some subpopulations (e.g., total EMR values for males ≥ 2% and EMR values for males aged 40-64 ≥ 1% in certain areas). We discuss these observations in light of the estimates of infection fatality rate (IFR) and eventual population fatality rate (PFR) made early in the course of the pandemic.

SARS-CoV-2 reinfections during the first three major COVID-19 waves in Bulgaria.

BACKGROUND: The COVID-19 pandemic has had a devastating impact on the world over the past two years (2020-2021). One of the key questions about its future trajectory is the protection from subsequent infections and disease conferred by a previous infection, as the SARS-CoV-2 virus belongs to the coronaviruses, a group of viruses the members of which are known for their ability to reinfect convalescent individuals. Bulgaria, with high rates of previous infections combined with low vaccination rates and an elderly population, presents a somewhat unique context to study this question. METHODS: We use detailed governmental data on registered COVID-19 cases to evaluate the incidence and outcomes of COVID-19 reinfections in Bulgaria in the period between March 2020 and early December 2021. RESULTS: For the period analyzed, a total of 4,106 cases of individuals infected more than once were observed, including 31 cases of three infections and one of four infections. The number of reinfections increased dramatically during the Delta variant-driven wave of the pandemic towards the end of 2021. We observe a moderate reduction of severe outcomes (hospitalization and death) in reinfections relative to primary infections, and a more substantial reduction of severe outcomes in breakthrough infections in vaccinated individuals. CONCLUSIONS: In the available datasets from Bulgaria, prior infection appears to provide some protection from severe outcomes, but to a lower degree than the reduction in severity of breakthrough infections in the vaccinated compared to primary infections in the unvaccinated.

The demographic and geographic impact of the COVID pandemic in Bulgaria and Eastern Europe in 2020.

The COVID-19 pandemic followed a unique trajectory in Eastern Europe compared to other heavily affected regions, with most countries there only experiencing a major surge of cases and deaths towards the end of 2020 after a relatively uneventful first half of the year. However, the consequences of that surge have not received as much attention as the situation in Western countries. Bulgaria, even though it has been one of the most heavily affected countries, has been one of those neglected cases. We use mortality and mobility data from Eurostat, official governmental and other sources to examine the development and impact of the COVID-19 pandemic in Bulgaria and other European countries. We find a very high level of excess mortality in Eastern European countries measured by several metrics including excess mortality rate (EMR), P-scores, potential years of life lost (PYLL) and its age standardised version (ASYR), and working years of life lost (WYLL). By the last three metrics Eastern Europe emerges as the hardest hit region by the pandemic in Europe in 2020. With a record EMR at ~0.27% and a strikingly large and mostly unique to it mortality rate in the working age (15-64 years) demographics, Bulgaria emerges as one of the most affected countries in Eastern Europe. The high excess mortality in Bulgaria correlates with insufficient intensity of testing, with delayed imposition of "lockdown" measures, and with high prevalence of cardiovascular diseases. We also find major geographic and demographic disparities within the country, with considerably lower mortality observed in major cities relative to more remote areas (likely due to disparities in the availability of medical resources). Analysis of the course of the epidemic revealed that individual mobility measures were predictive of the eventual decline in cases and deaths. However, while mobility declined as a result of the imposition of a lockdown, it already trended downwards before such measures were introduced, which resulted in a reduction of deaths independent of the effect of restrictions. Large excess mortality and high numbers of potential years of life lost are observed as a result of the COVID pandemic in Bulgaria, as well as in several other countries in Eastern Europe. Significant delays in the imposition of stringent mobility-reducing measures combined with a lack of medical resources likely caused a substantial loss of life, including in the working age population.

The chromatin organization of a chlorarachniophyte nucleomorph genome.

BACKGROUND: Nucleomorphs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features. RESULTS: In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain. CONCLUSIONS: We provide the first study of a nucleomorph genome using modern functional genomic tools, and derive numerous novel insights into the physical and functional organization of these unique genomes.