Genomic Nucleosome Organization Reconstituted with Pure Proteins.

Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.

RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.

Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters.

Genome-wide, little is understood about how proteins organize at inducible promoters before and after induction and to what extent inducible and constitutive architectures depend on cofactors. We report that sequence-specific transcription factors and their tethered cofactors (e.g., SAGA [Spt-Ada-Gcn5-acetyltransferase], Mediator, TUP, NuA4, SWI/SNF, and RPD3-L) are generally bound to promoters prior to induction ("poised"), rather than recruited upon induction, whereas induction recruits the preinitiation complex (PIC) to DNA. Through depletion and/or deletion experiments, we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 acetylates +1 nucleosomes there. When inducible promoters are poised, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. When induced, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID (transcription factor II-D) to stably associate, rather than creating a completely TFIID-independent PIC, as generally thought. These findings suggest that inducible systems, where present, are integrated with constitutive systems.

Understanding nucleosome dynamics and their links to gene expression and DNA replication.

Advances in genomics technology have provided the means to probe myriad chromatin interactions at unprecedented spatial and temporal resolution. This has led to a profound understanding of nucleosome organization within the genome, revealing that nucleosomes are highly dynamic. Nucleosome dynamics are governed by a complex interplay of histone composition, histone post-translational modifications, nucleosome occupancy and positioning within chromatin, which are influenced by numerous regulatory factors, including general regulatory factors, chromatin remodellers, chaperones and polymerases. It is now known that these dynamics regulate diverse cellular processes ranging from gene transcription to DNA replication and repair.

Subnucleosomal structures and nucleosome asymmetry across a genome.

Genes are packaged into nucleosomal arrays, each nucleosome typically having two copies of histones H2A, H2B, H3, and H4. Histones have distinct posttranslational modifications, variant isoforms, and dynamics. Whether each histone copy within a nucleosome has distinct properties, particularly in relation to the direction of transcription, is unknown. Here we use chromatin immunoprecipitation-exonuclease (ChIP-exo) to resolve the organization of individual histones on a genomic scale. We detect widespread subnucleosomal structures in dynamic chromatin, including what appear to be half-nucleosomes consisting of one copy of each histone. We also detect interactions of H3 tails with linker DNA between nucleosomes, which may be negatively regulated by methylation of H3K36. Histone variant H2A.Z is enriched on the promoter-distal half of the +1 nucleosome, whereas H2BK123 ubiquitylation and H3K9 acetylation are enriched on the promoter-proximal half in a transcription-linked manner. Subnucleosome asymmetries might serve as molecular beacons that guide transcription.

SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.

SWR-C/SWR1 and INO80 are multisubunit complexes that catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosome at the start of genes. How they target and engage these +1 nucleosomes is unclear. Using ChIP-exo, we identified the subnucleosomal placement of 20 of their subunits across the yeast genome. The Swc2 subunit of SWR-C bound a narrowly defined region in the adjacent nucleosome-free region (NFR), where it positioned the Swr1 subunit over one of two sites of H2A.Z deposition at +1. The genomic binding maps suggest that many subunits have a rather plastic organization that allows subunits to exchange between the two complexes. One outcome of promoting H2A/H2A.Z exchange was an enhanced turnover of entire nucleosomes, thereby creating dynamic chromatin at the start of genes. Our findings provide unifying concepts on how these two opposing chromatin remodeling complexes function selectively at the +1 nucleosome of nearly all genes.

Genome-wide nucleosome specificity and directionality of chromatin remodelers.

How chromatin remodelers cooperate to organize nucleosomes around the start and end of genes is not known. We determined the genome-wide binding of remodeler complexes SWI/SNF, RSC, ISW1a, ISW1b, ISW2, and INO80 to individual nucleosomes in Saccharomyces, and determined their functional contributions to nucleosome positioning through deletion analysis. We applied ultra-high-resolution ChIP-exo mapping to Isw2 to determine its subnucleosomal orientation and organization on a genomic scale. Remodelers interacted with selected nucleosome positions relative to the start and end of genes and produced net directionality in moving nucleosomes either away or toward nucleosome-free regions at the 5' and 3' ends of genes. Isw2 possessed a subnucleosomal organization in accord with biochemical and crystallographic-based models that place its linker binding region within promoters and abutted against Reb1-bound locations. Together, these findings reveal a coordinated position-specific approach taken by remodelers to organize genic nucleosomes into arrays.

A high-resolution protein architecture of the budding yeast genome.

The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.

High-resolution genome-wide mapping of the primary structure of chromatin.

The genomic organization of chromatin is increasingly recognized as a key regulator of cell behavior, but deciphering its regulation mechanisms requires detailed knowledge of chromatin's primary structure-the assembly of nucleosomes throughout the genome. This Primer explains the principles for mapping and analyzing the primary organization of chromatin on a genomic scale. After introducing chromatin organization and its impact on gene regulation and human health, we then describe methods that detect nucleosome positioning and occupancy levels using chromatin immunoprecipitation in combination with deep sequencing (ChIP-Seq), a strategy that is now straightforward and cost efficient. We then explore current strategies for converting the sequence information into knowledge about chromatin, an exciting challenge for biologists and bioinformaticians.

Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.

Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA-binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.

Phase separation directs ubiquitination of gene-body nucleosomes.

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.

Genome-wide promoter assembly in E. coli measured at single-base resolution.

When detected at single-base-pair resolution, the genome-wide location, occupancy level, and structural organization of DNA-binding proteins provide mechanistic insights into genome regulation. Here we use ChIP-exo to provide a near-base-pair resolution view of the epigenomic organization of the Escherichia coli transcription machinery and nucleoid structural proteins at the time when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We examined the site specificity of three sigma factors (RpoD/σ70, RpoH/σ32, and RpoN/σ54), RNA polymerase (RNAP or RpoA, -B, -C), and two nucleoid proteins (Fis and IHF). We suggest that DNA shape at the flanks of cognate motifs helps drive site specificity. We find that although RNAP and sigma factors occupy active cognate promoters, RpoH and RpoN can occupy quiescent promoters without the presence of RNAP. Thus, promoter-bound sigma factors can be triggered to recruit RNAP by a mechanism that is distinct from an obligatory cycle of free sigma binding RNAP followed by promoter binding. These findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape, and further define mechanistic steps in regulated genome-wide assembly of RNAP at promoters in E. coli.

GenoPipe: identifying the genotype of origin within (epi)genomic datasets.

Confidence in experimental results is critical for discovery. As the scale of data generation in genomics has grown exponentially, experimental error has likely kept pace despite the best efforts of many laboratories. Technical mistakes can and do occur at nearly every stage of a genomics assay (i.e. cell line contamination, reagent swapping, tube mislabelling, etc.) and are often difficult to identify post-execution. However, the DNA sequenced in genomic experiments contains certain markers (e.g. indels) encoded within and can often be ascertained forensically from experimental datasets. We developed the Genotype validation Pipeline (GenoPipe), a suite of heuristic tools that operate together directly on raw and aligned sequencing data from individual high-throughput sequencing experiments to characterize the underlying genome of the source material. We demonstrate how GenoPipe validates and rescues erroneously annotated experiments by identifying unique markers inherent to an organism's genome (i.e. epitope insertions, gene deletions and SNPs).

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, 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. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). 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 high-confidence 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 were 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 show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

Tail and Kinase Modules Differently Regulate Core Mediator Recruitment and Function In Vivo.

Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles.

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation.

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

STENCIL: A web templating engine for visualizing and sharing life science datasets.

The ability to aggregate experimental data analysis and results into a concise and interpretable format is a key step in evaluating the success of an experiment. This critical step determines baselines for reproducibility and is a key requirement for data dissemination. However, in practice it can be difficult to consolidate data analyses that encapsulates the broad range of datatypes available in the life sciences. We present STENCIL, a web templating engine designed to organize, visualize, and enable the sharing of interactive data visualizations. STENCIL leverages a flexible web framework for creating templates to render highly customizable visual front ends. This flexibility enables researchers to render small or large sets of experimental outcomes, producing high-quality downloadable and editable figures that retain their original relationship to the source data. REST API based back ends provide programmatic data access and supports easy data sharing. STENCIL is a lightweight tool that can stream data from Galaxy, a popular bioinformatic analysis web platform. STENCIL has been used to support the analysis and dissemination of two large scale genomic projects containing the complete data analysis for over 2,400 distinct datasets. Code and implementation details are available on GitHub: https://github.com/CEGRcode/stencil.