Search

Torsional stress in chromatin plays a fundamental role in cellular functions, influencing key processes such as transcription, replication, and chromatin organization. Transcription and other processes may generate and be regulated by torsional stress. In the genome, the interplay of these processes creates complicated patterns of both positive (+) and negative (-) torsion. However, a challenge in generating an accurate torsion map is determining the zero-torsion baseline signal, which is conflated with chromatin accessibility. Here, we introduce a high-resolution method based on the intercalator trimethylpsoralen (TMP) to address this challenge. We describe a method to establish the zero-torsion baseline while preserving the chromatin state of the genome of S. cerevisiae . This approach enables both high-resolution mapping of accessibility and torsional stress in chromatin in the cell. Our analysis shows transcription-generated torsional domains consistent with the twin-supercoiled-domain model of transcription and suggests a role for torsional stress in recruiting topoisomerases and in regulating 3D genome architecture via cohesin. Significantly, we reveal that insulator sequence-specific transcription factors decouple torsion between divergent promoters, whereas torsion spreads between divergent promoters lacking these factors, suggesting that torsion serves as a regulatory mechanism in these regions. Although insulators are known to decouple gene expression, our finding provides a physical explanation of how such decoupling may occur. This new method provides a potential path forward for using TMP to measure torsional stress in the genome without the confounding contribution of accessibility in chromatin., Competing Interests: Competing interest statement The authors declare no competing financial interests.

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes., Competing Interests: Declaration of interests B.F.P. is an owner of and has a financial interest in Peconic, which uses the ChIP-exo technology (US Patent 20100323361A1) implemented in this study and could potentially benefit from the outcomes of this research., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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)., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of interests B.F.P. is an owner of and has a financial interest in Peconic, which uses the ChIP-exo technology (U.S. Patent 20100323361A1) implemented in this study and could potentially benefit from the outcomes of this research., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The DNA-binding activities of transcription factors (TFs) are influenced by both intrinsic sequence preferences and extrinsic interactions with cell-specific chromatin landscapes and other regulatory proteins. Disentangling the roles of these binding determinants remains challenging. For example, the FoxA subfamily of Forkhead domain (Fox) TFs are known pioneer factors that can bind to relatively inaccessible sites during development. Yet FoxA TF binding also varies across cell types, pointing to a combination of intrinsic and extrinsic forces guiding their binding. While other Forkhead domain TFs are often assumed to have pioneering abilities, how sequence and chromatin features influence the binding of related Fox TFs has not been systematically characterized. Here, we present a principled approach to compare the relative contributions of intrinsic DNA sequence preference and cell-specific chromatin environments to a TF's DNA-binding activities. We apply our approach to investigate how a selection of Fox TFs (FoxA1, FoxC1, FoxG1, FoxL2, and FoxP3) vary in their binding specificity. We over-express the selected Fox TFs in mouse embryonic stem cells, which offer a platform to contrast each TF's binding activity within the same preexisting chromatin background. By applying a convolutional neural network to interpret the Fox TF binding patterns, we evaluate how sequence and preexisting chromatin features jointly contribute to induced TF binding. We demonstrate that Fox TFs bind different DNA targets, and drive differential gene expression patterns, even when induced in identical chromatin settings. Despite the association between Forkhead domains and pioneering activities, the selected Fox TFs display a wide range of affinities for preexiting chromatin states. Using sequence and chromatin feature attribution techniques to interpret the neural network predictions, we show that differential sequence preferences combined with differential abilities to engage relatively inaccessible chromatin together explain Fox TF binding patterns at individual sites and genome-wide., Competing Interests: Competing interests: B.F.P. has a financial interest in Peconic, LLC, which offers the ChIP–exo technology (US Patent 20100323361A1) implemented herein as a commercial service and could potentially benefit from the outcomes of this research. The remaining authors declare no competing interests.

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).

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., (© 2022 Mittal et al.; Published by Cold Spring Harbor Laboratory Press.)

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/σ 70 , RpoH/σ 32 , and RpoN/σ 54 ), 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 ., (© 2022 John et al.; Published by Cold Spring Harbor Laboratory Press.)

Reproducibility is a significant challenge in (epi)genomic research due to the complexity of experiments composed of traditional biochemistry and informatics. Recent advances have exacerbated this as high-throughput sequencing data is generated at an unprecedented pace. Here, we report the development of a Platform for Epi-Genomic Research (PEGR), a web-based project management platform that tracks and quality controls experiments from conception to publication-ready figures, compatible with multiple assays and bioinformatic pipelines. It supports rigor and reproducibility for biochemists working at the bench, while fully supporting reproducibility and reliability for bioinformaticians through integration with the Galaxy platform., (© 2022. The Author(s).)

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: BFP has a financial interest in Peconic, LLC, which utilizes the ChIP-exo technology mentioned in this study and could potentially benefit from the outcomes of this research.

In Saccharomyces cerevisiae , RNA polymerase II (Pol II) selects transcription start sites (TSSs) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 bp downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow., Competing Interests: TZ, IV, WL, SB, BN, CK No competing interests declared, BP BFP has a financial interest in Peconic, LLC, which utilizes the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research., (© 2021, Zhao et al.)

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., (© 2021 Lai et al.; Published by Cold Spring Harbor Laboratory Press.)

Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID), and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish a transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BFP has a financial interest in Peconic, LLC, which uses the ChIP-exo technology implemented in this study and could potentially benefit from the outcomes of this research., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

In multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30-50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert., Competing Interests: Declaration of interests B.F.P. is an owner of and has a financial interest in Peconic, which uses the ChIP-exo technology (U.S. Patent 20100323361A1) implemented in this study and could potentially benefit from the outcomes of this research. All other authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nuclear actin has been elusive due to the lack of knowledge about molecular mechanisms. From actin-containing chromatin remodeling complexes, we discovered an arginine mono-methylation mark on an evolutionarily conserved R256 residue of actin (R256me1). Actin R256 mutations in yeast affect nuclear functions and cause diseases in human. Interestingly, we show that an antibody specific for actin R256me1 preferentially stains nuclear actin over cytoplasmic actin in yeast, mouse, and human cells. We also show that actin R256me1 is regulated by protein arginine methyl transferase-5 (PRMT5) in HEK293 cells. A genome-wide survey of actin R256me1 mark provides a landscape for nuclear actin correlated with transcription. Further, gene expression and protein interaction studies uncover extensive correlations between actin R256me1 and active transcription. The discovery of actin R256me1 mark suggests a fundamental mechanism to distinguish nuclear actin from cytoplasmic actin through post-translational modification (PTM) and potentially implicates an actin PTM mark in transcription and human diseases., Competing Interests: Declaration of Interests B.F.P. has a financial interest in Piconic, LLC, which uses the ChIP-exo technology implemented in this study, and could potentially benefit from the outcomes of this research., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

There has been a rapid development in genome sequencing, including high-throughput next generation sequencing (NGS) technologies, automation in biological experiments, new bioinformatics tools and utilization of high-performance computing and cloud computing. ChIP-based NGS technologies, e.g. ChIP-seq and ChIP-exo, are widely used to detect the binding sites of DNA-interacting proteins in the genome and help us to have a deeper mechanistic understanding of genomic regulation. As sequencing data is generated at an unprecedented pace from the ChIP-based NGS pipelines, there is an urgent need for a metadata management system. To meet this need, we developed the Platform for Eukaryotic Genomic Regulation (PEGR), a web service platform that logs metadata for samples and sequencing experiments, manages the data processing workflows, and provides reporting and visualization. PEGR links together people, samples, protocols, DNA sequencers and bioinformatics computation. With the help of PEGR, scientists can have a more integrated understanding of the sequencing data and better understand the scientific mechanisms of genomic regulation. In this paper, we present the architecture and the major functionalities of PEGR. We also share our experience in developing this application and discuss the future directions.

Background: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function., Results: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model., Conclusions: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

Regulatory proteins can employ multiple direct and indirect modes of interaction with the genome. The ChIP-exo mixture model (ChExMix) provides a principled approach to detecting multiple protein-DNA interaction modes in a single ChIP-exo experiment. ChExMix discovers and characterizes binding event subtypes in ChIP-exo data by leveraging both protein-DNA cross-linking signatures and DNA motifs. In this study, we present a summary of the major features and applications of ChExMix. We demonstrate that ChExMix does not require high-resolution protein-DNA binding assay data to detect binding event subtypes. Specifically, we apply ChExMix to analyze 393 ChIP-seq data profiles in K562 cells. Similar binding event subtypes are discovered across multiple proteins, suggesting the existence of colocalized regulatory protein modules that are recruited to DNA through a particular sequence-specific transcription factor. Our results thus suggest that ChExMix can characterize protein-DNA binding interaction modes using data from multiple types of protein-DNA interaction assays.

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 cycle 1 . Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II 2 , the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation 3 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.

Motivation: Regulatory proteins associate with the genome either by directly binding cognate DNA motifs or via protein-protein interactions with other regulators. Each recruitment mechanism may be associated with distinct motifs and may also result in distinct characteristic patterns in high-resolution protein-DNA binding assays. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5' → 3' exonuclease digestion. Since different regulatory complexes will result in different protein-DNA crosslinking signatures, analysis of ChIP-exo tag enrichment patterns should enable detection of multiple protein-DNA binding modes for a given regulatory protein. However, current ChIP-exo analysis methods either treat all binding events as being of a uniform type or rely on motifs to cluster binding events into subtypes., Results: To systematically detect multiple protein-DNA interaction modes in a single ChIP-exo experiment, we introduce the ChIP-exo mixture model (ChExMix). ChExMix probabilistically models the genomic locations and subtype memberships of binding events using both ChIP-exo tag distribution patterns and DNA motifs. We demonstrate that ChExMix achieves accurate detection and classification of binding event subtypes using in silico mixed ChIP-exo data. We further demonstrate the unique analysis abilities of ChExMix using a collection of ChIP-exo experiments that profile the binding of key transcription factors in MCF-7 cells. In these data, ChExMix identifies possible recruitment mechanisms of FoxA1 and ERα, thus demonstrating that ChExMix can effectively stratify ChIP-exo binding events into biologically meaningful subtypes., Availability and Implementation: ChExMix is available from https://github.com/seqcode/chexmix., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating complex roles in cell and systems biology. Species differences in CAR's effector pathways remain poorly understood, including its role in regulating liver tumor promotion. We developed transgenic mouse models to assess genome-wide binding of mouse and human CAR, following receptor activation in liver with direct ligands and with phenobarbital, an indirect CAR activator. Genomic interaction profiles were integrated with transcriptional and biological pathway analyses. Newly identified CAR target genes included Gdf15 and Foxo3, important regulators of the carcinogenic process. Approximately 1000 genes exhibited differential binding interactions between mouse and human CAR, including the proto-oncogenes, Myc and Ikbke, which demonstrated preferential binding by mouse CAR as well as mouse CAR-selective transcriptional enhancement. The ChIP-exo analyses also identified distinct binding motifs for the respective mouse and human receptors. Together, the results provide new insights into the important roles that CAR contributes as a key modulator of numerous signaling pathways in mammalian organisms, presenting a genomic context that specifies species variation in biological processes under CAR's control, including liver cell proliferation and tumor promotion.

ChIP-seq and ChIP-exo identify where proteins bind along any genome in vivo. Although ChIP-seq is widely adopted in academic research, it has inherently high noise. In contrast, ChIP-exo has relatively low noise and achieves near-base pair resolution. Consequently, and unlike other genomic assays, ChIP-exo provides structural information on genome-wide binding proteins. Construction of ChIP-exo libraries is technically difficult. Here we describe greatly simplified ChIP-exo methods, each with use-specific advantages. This is achieved through assay optimization and use of Tn5 tagmentation and/or single-stranded DNA ligation. Greater library yields, lower processing time, and lower costs are achieved. In comparing assays, we reveal substantial limitations in other ChIP-based assays. Importantly, the new ChIP-exo assays allow high-resolution detection of some protein-DNA interactions in organs and in as few as 27,000 cells. It is suitable for high-throughput parallelization. The simplicity of ChIP-exo now makes it a highly appropriate substitute for ChIP-seq, and for broader adoption.

General regulatory factors (GRFs), such as Reb1, Abf1, Rap1, Mcm1, and Cbf1, positionally organize yeast chromatin through interactions with a core consensus DNA sequence. It is assumed that sequence recognition via direct base readout suffices for specificity and that spurious nonfunctional sites are rendered inaccessible by chromatin. We tested these assumptions through genome-wide mapping of GRFs in vivo and in purified biochemical systems at near-base pair (bp) resolution using several ChIP-exo-based assays. We find that computationally predicted DNA shape features (e.g., minor groove width, helix twist, base roll, and propeller twist) that are not defined by a unique consensus sequence are embedded in the nonunique portions of GRF motifs and contribute critically to sequence-specific binding. This dual source specificity occurs at GRF sites in promoter regions where chromatin organization starts. Outside of promoter regions, strong consensus sites lack the shape component and consequently lack an intrinsic ability to bind cognate GRFs, without regard to influences from chromatin. However, sites having a weak consensus and low intrinsic affinity do exist in these regions but are rendered inaccessible in a chromatin environment. Thus, GRF site-specificity is achieved through integration of favorable DNA sequence and shape readouts in promoter regions and by chromatin-based exclusion from fortuitous weak sites within gene bodies. This study further revealed a severe G/C nucleotide cross-linking selectivity inherent in all formaldehyde-based ChIP assays, which includes ChIP-seq. However, for most tested proteins, G/C selectivity did not appreciably affect binding site detection, although it does place limits on the quantitativeness of occupancy levels., (© 2018 Rossi et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Eukaryotic transcription is regulated through two complexes, the general transcription factor IID (TFIID) and the coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA). Recent findings confirm that both TFIID and SAGA contribute to the synthesis of nearly all transcripts and are recruited genome-wide in yeast. However, how this broad recruitment confers selectivity under specific conditions remains an open question., Results: Here we find that the SAGA/TREX-2 subunit Sus1 associates with upstream regulatory regions of many yeast genes and that heat shock drastically changes Sus1 binding. While Sus1 binding to TFIID-dominated genes is not affected by temperature, its recruitment to SAGA-dominated genes and RP genes is significantly disturbed under heat shock, with Sus1 relocated to environmental stress-responsive genes in these conditions. Moreover, in contrast to recent results showing that SAGA deubiquitinating enzyme Ubp8 is dispensable for RNA synthesis, genomic run-on experiments demonstrate that Sus1 contributes to synthesis and stability of a wide range of transcripts., Conclusions: Our study provides support for a model in which SAGA/TREX-2 factor Sus1 acts as a global transcriptional regulator in yeast but has differential activity at yeast genes as a function of their transcription rate or during stress conditions.

Gene expression is controlled by a variety of proteins that interact with the genome. Their precise organization and mechanism of action at every promoter remains to be worked out. To better understand the physical interplay among genome-interacting proteins, we examined the temporal binding of a functionally diverse subset of these proteins: nucleosomes (H3), H2AZ (Htz1), SWR (Swr1), RSC (Rsc1, Rsc3, Rsc58, Rsc6, Rsc9, Sth1), SAGA (Spt3, Spt7, Ubp8, Sgf11), Hsf1, TFIID (Spt15/TBP and Taf1), TFIIB (Sua7), TFIIH (Ssl2), FACT (Spt16), Pol II (Rpb3), and Pol II carboxyl-terminal domain (CTD) phosphorylation at serines 2, 5, and 7. They were examined under normal and acute heat shock conditions, using the ultrahigh resolution genome-wide ChIP-exo assay in Saccharomyces cerevisiae Our findings reveal a precise positional organization of proteins bound at most genes, some of which rapidly reorganize within minutes of heat shock. This includes more precise positional transitions of Pol II CTD phosphorylation along the 5' ends of genes than previously seen. Reorganization upon heat shock includes colocalization of SAGA with promoter-bound Hsf1, a change in RSC subunit enrichment from gene bodies to promoters, and Pol II accumulation within promoter/+1 nucleosome regions. Most of these events are widespread and not necessarily coupled to changes in gene expression. Together, these findings reveal protein-genome interactions that are robustly reprogrammed in precise and uniform ways far beyond what is elicited by changes in gene expression., (© 2018 Vinayachandran et al.; Published by Cold Spring Harbor Laboratory Press.)

The Ccr4 (carbon catabolite repression 4)-Not complex is a major regulator of stress responses that controls gene expression at multiple levels, from transcription to mRNA decay. Ccr4, a "core" subunit of the complex, is the main cytoplasmic deadenylase in Saccharomyces cerevisiae ; however, its mRNA targets have not been mapped on a genome-wide scale. Here, we describe a genome-wide approach, RNA immunoprecipitation (RIP) high-throughput sequencing (RIP-seq), to identify the RNAs bound to Ccr4, and two proteins that associate with it, Dhh1 and Puf5 All three proteins were preferentially bound to lowly abundant mRNAs, most often at the 3' end of the transcript. Furthermore, Ccr4, Dhh1, and Puf5 are recruited to mRNAs that are targeted by other RNA-binding proteins that promote decay and mRNA transport, and inhibit translation. Although Ccr4-Not regulates mRNA transcription and decay, Ccr4 recruitment to mRNAs correlates better with decay rates, suggesting it imparts greater control over transcript abundance through decay. Ccr4-enriched mRNAs are refractory to control by the other deadenylase complex in yeast, Pan2/3, suggesting a division of labor between these deadenylation complexes. Finally, Ccr4 and Dhh1 associate with mRNAs whose abundance increases during nutrient starvation, and those that fluctuate during metabolic and oxygen consumption cycles, which explains the known genetic connections between these factors and nutrient utilization and stress pathways., (Copyright © 2018 Miller et al.)

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.

Transcription of protein-coding and noncoding DNA occurs pervasively throughout the mammalian genome. Their sites of initiation are generally inferred from transcript 5' ends and are thought to be either locally dispersed or focused. How these two modes of initiation relate is unclear. Here, we apply permanganate treatment and chromatin immunoprecipitation (PIP-seq) of initiation factors to identify the precise location of melted DNA separately associated with the preinitiation complex (PIC) and the adjacent paused complex (PC). This approach revealed the two known modes of transcription initiation. However, in contrast to prevailing views, they co-occurred within the same promoter region: initiation originating from a focused PIC, and broad nucleosome-linked initiation. PIP-seq allowed transcriptional orientation of Pol II to be determined, which may be useful near promoters where sufficient sense/anti-sense transcript mapping information is lacking. PIP-seq detected divergently oriented Pol II at both coding and noncoding promoters, as well as at enhancers. Their occupancy levels were not necessarily coupled in the two orientations. DNA sequence and shape analysis of initiation complex sites suggest that both sequence and shape contribute to specificity, but in a context-restricted manner. That is, initiation sites have the locally "best" initiator (INR) sequence and/or shape. These findings reveal a common core to pervasive Pol II initiation throughout the human genome., (© 2017 Lai and Pugh; Published by Cold Spring Harbor Laboratory Press.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nap1 is a histone chaperone involved in the nuclear import of H2A-H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A-H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1-mediated H2A-H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A-H2B heterodimer. Oligomerization of the Nap1-H2A-H2B complex results in burial of surfaces required for deposition of H2A-H2B into nucleosomes. Chromatin immunoprecipitation-exonuclease (ChIP-exo) analysis shows that Nap1 is required for H2A-H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1-mediated H2A-H2B deposition and nucleosome assembly., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A repertoire of transcription initiation factors engage the core promoter of mRNA genes to recruit RNA polymerase (Pol) II to initiate transcription, yet their precise spatial organization remains unclear. Using ChIP-exo, here we detail the interactions and genomic organization of initiation factors TBP, TFIIB, and Pol II at mRNA genes and within CpG islands. We find that when Pol II moves into a transcriptionally paused state, TBP/TFIIB remain at the promoter. We show that TBP and TFIIB bound to the core promoter at two separate, resolvable locations that coincided with sites of divergent transcription initiation. We also examine the precise binding of TBP at Pol III transcribed tRNA genes. We find that TBP crosslinked to tRNA genes in a similar manner as at Pol II transcribed genes. This comprehensive and high resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events humans at Pol II coding and Pol III transcribed tRNA genes.

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Erythroid development and differentiation from multiprogenitor cells into red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, cooperate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are juxtaposed along the DNA and their cognate DNA binding site across the mouse genome remains unclear. We applied high-resolution ChIP-exo (chromatin immunoprecipitation followed by 5'-to-3' exonuclease treatment and then massively parallel DNA sequencing) to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak pairing, were used to determine high-confidence binding locations by ChIP-exo. We identified ∼10,000 GATA1 and ∼15,000 TAL1 locations, which were essentially confirmed by ChIP-seq (chromatin immunoprecipitation followed by massively parallel DNA sequencing). Of these, ∼4,000 locations were bound by both GATA1 and TAL1. About three-quarters of them were tightly linked to a partial E-box located 7 or 8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs that reflect their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7 or 8 bp upstream of a WGATAA motif across thousands of genomic locations., (Copyright © 2015 Han et al.)

The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences., (© 2015 Reja et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We previously reported that a pan-PAD inhibitor, YW3-56, activates p53 target genes to inhibit cancer growth. However, the p53-independent anticancer activity and molecular mechanisms of YW3-56 remain largely elusive. Here, gene expression analyses found that ATF4 target genes involved in endoplasmic reticulum (ER) stress response were activated by YW3-56. Depletion of ATF4 greatly attenuated YW3-56-mediated activation of the mTORC1 regulatory genes SESN2 and DDIT4. Using the ChIP-exo method, high-resolution genomic binding sites of ATF4 and CEBPB responsive to YW3-56 treatment were generated. In human breast cancer cells, YW3-56-mediated cell death features mitochondria depletion and autophagy perturbation. Moreover, YW3-56 treatment effectively inhibits the growth of triple-negative breast cancer xenograft tumors in nude mice. Taken together, we unveiled the anticancer mechanisms and therapeutic potentials of the pan-PAD inhibitor YW3-56., (©2015 American Association for Cancer Research.)

Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA cross-linking patterns by combining chromatin immunoprecipitation (ChIP) with 5' → 3' exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATAC-seq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The human genome is pervasively transcribed, yet only a small fraction is coding. Here we address whether this non-coding transcription arises at promoters, and detail the interactions of initiation factors TATA box binding protein (TBP), transcription factor IIB (TFIIB) and RNA polymerase (Pol) II. Using ChIP-exo (chromatin immunoprecipitation with lambda exonuclease digestion followed by high-throughput sequencing), we identify approximately 160,000 transcription initiation complexes across the human K562 genome, and more in other cancer genomes. Only about 5% associate with messenger RNA genes. The remainder associates with non-polyadenylated non-coding transcription. Regardless, Pol II moves into a transcriptionally paused state, and TBP and TFIIB remain at the promoter. Remarkably, the vast majority of locations contain the four core promoter elements- upstream TFIIB recognition element (BREu), TATA, downstream TFIIB recognition element (BREd), and initiator element (INR)-in constrained positions. All but the INR also reside at Pol III promoters, where TBP makes similar contacts. This comprehensive and high-resolution genome-wide detection of the initiation machinery produces a consolidated view of transcription initiation events from yeast to humans at Pol II/III TATA-containing/TATA-less coding and non-coding genes.