Uncovering uncharacterized binding of transcription factors from ATAC-seq footprinting data.

Transcription factors (TFs) are crucial epigenetic regulators, which enable cells to dynamically adjust gene expression in response to environmental signals. Computational procedures like digital genomic footprinting on chromatin accessibility assays such as ATACseq can be used to identify bound TFs in a genome-wide scale. This method utilizes short regions of low accessibility signals due to steric hindrance of DNA bound proteins, called footprints (FPs), which are combined with motif databases for TF identification. However, while over 1600 TFs have been described in the human genome, only ~ 700 of these have a known binding motif. Thus, a substantial number of FPs without overlap to a known DNA motif are normally discarded from FP analysis. In addition, the FP method is restricted to organisms with a substantial number of known TF motifs. Here we present DENIS (DE Novo motIf diScovery), a framework to generate and systematically investigate the potential of de novo TF motif discovery from FPs. DENIS includes functionality (1) to isolate FPs without binding motifs, (2) to perform de novo motif generation and (3) to characterize novel motifs. Here, we show that the framework rediscovers artificially removed TF motifs, quantifies de novo motif usage during an early embryonic development example dataset, and is able to analyze and uncover TF activity in organisms lacking canonical motifs. The latter task is exemplified by an investigation of a scATAC-seq dataset in zebrafish which covers different cell types during hematopoiesis.

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting.

Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.

The native cistrome and sequence motif families of the maize ear.

Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.

Genomic Footprinting Analyses from DNase-seq Data to Construct Gene Regulatory Networks.

Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.

Rapid and inexpensive preparation of genome-wide nucleosome footprints from model and non-model organisms.

MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

Using an L7Ae-Tethered, Hydroxyl Radical-Mediated Footprinting Strategy to Identify and Validate Kink-Turns in RNAs.

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.

In Vivo Footprinting Analysis in Trichoderma reesei.

The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo.

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.

ATAC-seq with unique molecular identifiers improves quantification and footprinting.

ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq .

Molecular Footprints of the Immune Assault on Pancreatic Beta Cells in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic disease caused by the selective destruction of the insulin-producing pancreatic beta cells by infiltrating immune cells. We presently evaluated the transcriptomic signature observed in beta cells in early T1D and compared it with the signatures observed following in vitro exposure of human islets to inflammatory or metabolic stresses, with the aim of identifying "footprints" of the immune assault in the target beta cells. We detected similarities between the beta cell signatures induced by cytokines present at different moments of the disease, i.e., interferon-α (early disease) and interleukin-1ß plus interferon-γ (later stages) and the beta cells from T1D patients, identifying biological process and signaling pathways activated during early and late stages of the disease. Among the first responses triggered on beta cells was an enrichment in antiviral responses, pattern recognition receptors activation, protein modification and MHC class I antigen presentation. During putative later stages of insulitis the processes were dominated by T-cell recruitment and activation and attempts of beta cells to defend themselves through the activation of anti-inflammatory pathways (i.e., IL10, IL4/13) and immune check-point proteins (i.e., PDL1 and HLA-E). Finally, we mined the beta cell signature in islets from T1D patients using the Connectivity Map, a large database of chemical compounds/drugs, and identified interesting candidates to potentially revert the effects of insulitis on beta cells.

CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites.

CUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control.

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

TRACE: transcription factor footprinting using chromatin accessibility data and DNA sequence.

Transcription is tightly regulated by cis-regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to understanding gene expression and whole regulatory networks within a cell. The standard approaches used for TFBS prediction, such as position weight matrices (PWMs) and chromatin immunoprecipitation followed by sequencing (ChIP-seq), are widely used but have their drawbacks, including high false-positive rates and limited antibody availability, respectively. Several computational footprinting algorithms have been developed to detect TFBSs by investigating chromatin accessibility patterns; however, these also have limitations. We have developed a footprinting method to predict TF footprints in active chromatin elements (TRACE) to improve the prediction of TFBS footprints. TRACE incorporates DNase-seq data and PWMs within a multivariate hidden Markov model (HMM) to detect footprint-like regions with matching motifs. TRACE is an unsupervised method that accurately annotates binding sites for specific TFs automatically with no requirement for pregenerated candidate binding sites or ChIP-seq training data. Compared with published footprinting algorithms, TRACE has the best overall performance with the distinct advantage of targeting multiple motifs in a single model.

Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells.

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.

The Ferric Uptake Regulator Represses Type VI Secretion System Function by Binding Directly to the clpV Promoter in Salmonella enterica Serovar Typhimurium.

Type VI secretion systems (T6SSs) are highly conserved and complex protein secretion systems that deliver effector proteins into eukaryotic hosts or other bacteria. T6SSs are regulated precisely by a variety of regulatory systems, which enables bacteria to adapt to varied environments. A T6SS within Salmonella pathogenicity island 6 (SPI-6) is activated during infection, and it contributes to the pathogenesis, as well as interbacterial competition, of Salmonella enterica serovar Typhimurium (S. Typhimurium). However, the regulation of the SPI-6 T6SS in S. Typhimurium is not well understood. In this study, we found that the SPI-6 T6SS core gene clpV was significantly upregulated in response to the iron-depleted condition and during infection. The global ferric uptake regulator (Fur) was shown to repress the clpV expression in the iron-replete medium. Moreover, electrophoretic mobility shift and DNase I footprinting assays revealed that Fur binds directly to the clpV promoter region at multiple sites spanning the transcriptional start site. We also observed that the relieving of Fur-mediated repression on clpV contributed to the interbacterial competition activity and pathogenicity of S. Typhimurium. These findings provide insights into the direct regulation of Fur in the expression and functional activity of SPI-6 T6SS in S. Typhimurium and thus help to elucidate the mechanisms of bacterial adaptability and virulence.

XL-DNase-seq: improved footprinting of dynamic transcription factors.

BACKGROUND: As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. RESULTS: Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. CONCLUSIONS: XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.

Identification of transcription factor binding sites using ATAC-seq.

Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) is a simple protocol for detection of open chromatin. Computational footprinting, the search for regions with depletion of cleavage events due to transcription factor binding, is poorly understood for ATAC-seq. We propose the first footprinting method considering ATAC-seq protocol artifacts. HINT-ATAC uses a position dependency model to learn the cleavage preferences of the transposase. We observe strand-specific cleavage patterns around transcription factor binding sites, which are determined by local nucleosome architecture. By incorporating all these biases, HINT-ATAC is able to significantly outperform competing methods in the prediction of transcription factor binding sites with footprints.

Early Parity Epigenetic Footprint of FOXA1 Gene Body in Normal Breast Tissue of Iranian Women

Background: Young age at first full-term pregnancy (FFTP) is an important factor in breast cancer risk reduction. It is postulated that this protective effect is the result of stable molecular signatures imprinted by physiological process of pregnancy, but the molecular mechanism of this protective role is unclear. The aim of the current study was to identify the effect of early FFTP on methylation status of FOXA1 gene body. FOXA1 is an essential transcription factor for mammary gland development and estrogen responsiveness of breast tissue. Methods: Fresh frozen normal breast tissues (n = 51) were collected from Iranian women who underwent cosmetic mammoplasty (27 nulliparous women and 24 parous women who have experienced first pregnancy before the age of 25). DNA was extracted and then methylated DNA immunoprecipitation (MeDIP) real-time PCR was used to assess FOXA1 gene body methylation. Results: Our results revealed that FOXA1 methylation level is significantly higher in early parous compared with nulliparous group (p = 0.041). Conclusion: Our study provides new hint about the association between early FFTP and epigenetic modifications within gene body of FOXA1 in normal breast tissue. More investigation is required for clarifying molecular mechanisms underlying this association in order to develop breast cancer prevention strategies.

Evaluation of Nrf2 with Exposure to Nanoparticles.

Transcription factor Nrf2, nuclear factor (erythroid-derived 2)-like 2, is considered a master regulator of redox homeostasis and plays a central role in antioxidant and anti-inflammatory defence. It has been largely reported that oxidative stress is implicated in nanoparticle-induced toxicity with the involvement of Nrf2. Several basic methods for Nrf2 evaluation with exposure to nanoparticles are described in this chapter including real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunofluorescence staining, electrophoretic mobility shift assay, DNase I footprinting, dimethylsulfate footprinting, protein pulse-chase analysis, and tert-butylhydroquinone treatment.

Structural interpretation of DNA-protein hydroxyl-radical footprinting experiments with high resolution using HYDROID.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.