Journal articles: 'DNase-seq'

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Liu, Yongjing, Liangyu Fu, Kerstin Kaufmann, Dijun Chen, and Ming Chen. "A practical guide for DNase-seq data analysis: from data management to common applications." Briefings in Bioinformatics 20, no. 5 (June 4, 2019): 1865–77. http://dx.doi.org/10.1093/bib/bby057.

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Abstract Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon. However, exploring DNase-seq data requires sophisticated downstream bioinformatics analyses. In this study, we first review computational methods for all of the major steps in DNase-seq data analysis, including experimental design, quality control, read alignment, peak calling, annotation of cis-regulatory elements, genomic footprinting and visualization. The challenges associated with each step are highlighted. Next, we provide a practical guideline and a computational pipeline for DNase-seq data analysis by integrating some of these tools. We also discuss the competing techniques and the potential applications of this pipeline for the analysis of analogous experimental data. Finally, we discuss the integration of DNase-seq with other functional genomics techniques.

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Sun, H., B. Qin, T. Liu, Q. Wang, J. Liu, J. Wang, X. Lin, et al. "CistromeFinder for ChIP-seq and DNase-seq data reuse." Bioinformatics 29, no. 10 (March 18, 2013): 1352–54. http://dx.doi.org/10.1093/bioinformatics/btt135.

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Zhong, Jianling, Kaixuan Luo, Peter S. Winter, Gregory E. Crawford, Edwin S. Iversen, and Alexander J. Hartemink. "Mapping nucleosome positions using DNase-seq." Genome Research 26, no. 3 (January 15, 2016): 351–64. http://dx.doi.org/10.1101/gr.195602.115.

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Gao, Weiwu, Wai Lim Ku, Lixia Pan, Jonathan Perrie, Tingting Zhao, Gangqing Hu, Yuzhang Wu, Jun Zhu, Bing Ni, and Keji Zhao. "Multiplex indexing approach for the detection of DNase I hypersensitive sites in single cells." Nucleic Acids Research 49, no. 10 (March 8, 2021): e56-e56. http://dx.doi.org/10.1093/nar/gkab102.

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Abstract Single cell chromatin accessibility assays reveal epigenomic variability at cis-regulatory elements among individual cells. We previously developed a single-cell DNase-seq assay (scDNase-seq) to profile accessible chromatin in a limited number of single cells. Here, we report a novel indexing strategy to resolve single-cell DNase hypersensitivity profiles based on bulk cell analysis. This new technique, termed indexing single-cell DNase sequencing (iscDNase-seq), employs the activities of terminal DNA transferase (TdT) and T4 DNA ligase to add unique cell barcodes to DNase-digested chromatin ends. By a three-layer indexing strategy, it allows profiling genome-wide DHSs for >15 000 single-cells in a single experiment. Application of iscDNase-seq to human white blood cells accurately revealed specific cell types and inferred regulatory transcription factors (TF) specific to each cell type. We found that iscDNase-seq detected DHSs with specific properties related to gene expression and conservation missed by scATAC-seq for the same cell type. Also, we found that the cell-to-cell variation in accessibility computed using iscDNase-seq data is significantly correlated with the cell-to-cell variation in gene expression. Importantly, this correlation is significantly higher than that between scATAC-seq and scRNA-seq, suggesting that iscDNase-seq data can better predict the cellular heterogeneity in gene expression compared to scATAC-seq. Thus, iscDNase-seq is an attractive alternative method for single-cell epigenomics studies.

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Nordström, Karl J. V., Florian Schmidt, Nina Gasparoni, Abdulrahman Salhab, Gilles Gasparoni, Kathrin Kattler, Fabian Müller, et al. "Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data." Nucleic Acids Research 47, no. 20 (October 4, 2019): 10580–96. http://dx.doi.org/10.1093/nar/gkz799.

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Abstract Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-seq, ATAC-seq and NOMe-seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However, we also find a high proportion of assay specific NDRs that are often ‘called’ by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-seq and DNase I-seq provide a superb high NDR calling rate for relatively low sequencing costs in comparison to NOMe-seq, NOMe-seq singles out for its genome-wide coverage allowing to not only detect NDRs but also endogenous DNA methylation and as we show here genome wide segmentation into heterochromatic B domains and local phasing of nucleosomes outside of NDRs. In summary, our comparisons strongly suggest to consider assay specific differences for the experimental design and for generalized and comparative functional interpretations.

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Taing, Len, Gali Bai, Clara Cousins, Paloma Cejas, Xintao Qiu, Zachary T. Herbert, Myles Brown, et al. "CHIPS: A Snakemake pipeline for quality control and reproducible processing of chromatin profiling data." F1000Research 10 (June 30, 2021): 517. http://dx.doi.org/10.12688/f1000research.52878.1.

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Motivation: The chromatin profile measured by ATAC-seq, ChIP-seq, or DNase-seq experiments can identify genomic regions critical in regulating gene expression and provide insights on biological processes such as diseases and development. However, quality control and processing chromatin profiling data involves many steps, and different bioinformatics tools are used at each step. It can be challenging to manage the analysis. Results: We developed a Snakemake pipeline called CHIPS (CHromatin enrIchment ProcesSor) to streamline the processing of ChIP-seq, ATAC-seq, and DNase-seq data. The pipeline supports single- and paired-end data and is flexible to start with FASTQ or BAM files. It includes basic steps such as read trimming, mapping, and peak calling. In addition, it calculates quality control metrics such as contamination profiles, polymerase chain reaction bottleneck coefficient, the fraction of reads in peaks, percentage of peaks overlapping with the union of public DNaseI hypersensitivity sites, and conservation profile of the peaks. For downstream analysis, it carries out peak annotations, motif finding, and regulatory potential calculation for all genes. The pipeline ensures that the processing is robust and reproducible. Availability: CHIPS is available at https://github.com/liulab-dfci/CHIPS.

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Koohy, Hashem, Thomas A. Down, Mikhail Spivakov, and Tim Hubbard. "A Comparison of Peak Callers Used for DNase-Seq Data." PLoS ONE 9, no. 5 (May 8, 2014): e96303. http://dx.doi.org/10.1371/journal.pone.0096303.

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Tarbell, Evan D., and Tao Liu. "HMMRATAC: a Hidden Markov ModeleR for ATAC-seq." Nucleic Acids Research 47, no. 16 (June 14, 2019): e91-e91. http://dx.doi.org/10.1093/nar/gkz533.

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Abstract ATAC-seq has been widely adopted to identify accessible chromatin regions across the genome. However, current data analysis still utilizes approaches initially designed for ChIP-seq or DNase-seq, without considering the transposase digested DNA fragments that contain additional nucleosome positioning information. We present the first dedicated ATAC-seq analysis tool, a semi-supervised machine learning approach named HMMRATAC. HMMRATAC splits a single ATAC-seq dataset into nucleosome-free and nucleosome-enriched signals, learns the unique chromatin structure around accessible regions, and then predicts accessible regions across the entire genome. We show that HMMRATAC outperforms the popular peak-calling algorithms on published human ATAC-seq datasets. We find that single-end sequenced or size-selected ATAC-seq datasets result in a loss of sensitivity compared to paired-end datasets without size-selection.

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Cho, Jin Sun, Ira L. Blitz, and Ken W. Y. Cho. "DNase-seq to Study Chromatin Accessibility in Early Xenopus tropicalis Embryos." Cold Spring Harbor Protocols 2019, no. 4 (August 21, 2018): pdb.prot098335. http://dx.doi.org/10.1101/pdb.prot098335.

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10

Wang, Jiayin, Liubin Chen, Xuanping Zhang, Yao Tong, and Tian Zheng. "OCRDetector: Accurately Detecting Open Chromatin Regions via Plasma Cell-Free DNA Sequencing Data." International Journal of Molecular Sciences 22, no. 11 (May 28, 2021): 5802. http://dx.doi.org/10.3390/ijms22115802.

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Open chromatin regions (OCRs) are special regions of the human genome that can be accessed by DNA regulatory elements. Several studies have reported that a series of OCRs are associated with mechanisms involved in human diseases, such as cancers. Identifying OCRs using ATAC-seq or DNase-seq is often expensive. It has become popular to detect OCRs from plasma cell-free DNA (cfDNA) sequencing data, because both the fragmentation modes of cfDNA and the sequencing coverage in OCRs are significantly different from those in other regions. However, it is a challenging computational problem to accurately detect OCRs from plasma cfDNA-seq data, as multiple factors—e.g., sequencing and mapping bias, insufficient read depth, etc.—often mislead the computational model. In this paper, we propose a novel bioinformatics pipeline, OCRDetector, for detecting OCRs from whole-genome cfDNA sequencing data. The pipeline calculates the window protection score (WPS) waveform and the cfDNA sequencing coverage. To validate the proposed pipeline, we compared the percentage overlap of our OCRs with those obtained by other methods. The experimental results show that 81% of the TSS regions of housekeeping genes are detected, and our results have obvious tissue specificity. In addition, the overlap percentage between our OCRs and the high-confidence OCRs obtained by ATAC-seq or DNase-seq is greater than 70%.

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Qin, B., M. Zhou, Y. Ge, L. Taing, T. Liu, Q. Wang, S. Wang, et al. "CistromeMap: a knowledgebase and web server for ChIP-Seq and DNase-Seq studies in mouse and human." Bioinformatics 28, no. 10 (April 11, 2012): 1411–12. http://dx.doi.org/10.1093/bioinformatics/bts157.

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Zhou, Weiqiang, Zhicheng Ji, Weixiang Fang, and Hongkai Ji. "Global prediction of chromatin accessibility using small-cell-number and single-cell RNA-seq." Nucleic Acids Research 47, no. 19 (August 20, 2019): e121-e121. http://dx.doi.org/10.1093/nar/gkz716.

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Abstract Conventional high-throughput genomic technologies for mapping regulatory element activities in bulk samples such as ChIP-seq, DNase-seq and FAIRE-seq cannot analyze samples with small numbers of cells. The recently developed low-input and single-cell regulome mapping technologies such as ATAC-seq and single-cell ATAC-seq (scATAC-seq) allow analyses of small-cell-number and single-cell samples, but their signals remain highly discrete or noisy. Compared to these regulome mapping technologies, transcriptome profiling by RNA-seq is more widely used. Transcriptome data in single-cell and small-cell-number samples are more continuous and often less noisy. Here, we show that one can globally predict chromatin accessibility and infer regulatory element activities using RNA-seq. Genome-wide chromatin accessibility predicted by RNA-seq from 30 cells can offer better accuracy than ATAC-seq from 500 cells. Predictions based on single-cell RNA-seq (scRNA-seq) can more accurately reconstruct bulk chromatin accessibility than using scATAC-seq. Integrating ATAC-seq with predictions from RNA-seq increases the power and value of both methods. Thus, transcriptome-based prediction provides a new tool for decoding gene regulatory circuitry in samples with limited cell numbers.

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Kolmykov, Semyon, Ivan Yevshin, Mikhail Kulyashov, Ruslan Sharipov, Yury Kondrakhin, Vsevolod J. Makeev, Ivan V. Kulakovskiy, Alexander Kel, and Fedor Kolpakov. "GTRD: an integrated view of transcription regulation." Nucleic Acids Research 49, no. D1 (November 24, 2020): D104—D111. http://dx.doi.org/10.1093/nar/gkaa1057.

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Abstract The Gene Transcription Regulation Database (GTRD; http://gtrd.biouml.org/) contains uniformly annotated and processed NGS data related to gene transcription regulation: ChIP-seq, ChIP-exo, DNase-seq, MNase-seq, ATAC-seq and RNA-seq. With the latest release, the database has reached a new level of data integration. All cell types (cell lines and tissues) presented in the GTRD were arranged into a dictionary and linked with different ontologies (BRENDA, Cell Ontology, Uberon, Cellosaurus and Experimental Factor Ontology) and with related experiments in specialized databases on transcription regulation (FANTOM5, ENCODE and GTEx). The updated version of the GTRD provides an integrated view of transcription regulation through a dedicated web interface with advanced browsing and search capabilities, an integrated genome browser, and table reports by cell types, transcription factors, and genes of interest.

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Klare, William, Theerthankar Das, Amaye Ibugo, Edwina Buckle, Mike Manefield, and Jim Manos. "Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome." Antimicrobial Agents and Chemotherapy 60, no. 8 (May 9, 2016): 4539–51. http://dx.doi.org/10.1128/aac.02919-15.

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ABSTRACTPseudomonas aeruginosainfections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. WithinP. aeruginosabiofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pendingin vivostudies.

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Winter, D. R., L. Song, S. Mukherjee, T. S. Furey, and G. E. Crawford. "DNase-seq predicts regions of rotational nucleosome stability across diverse human cell types." Genome Research 23, no. 7 (May 8, 2013): 1118–29. http://dx.doi.org/10.1101/gr.150482.112.

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Moyerbrailean, Gregory A., Cynthia A. Kalita, Chris T. Harvey, Xiaoquan Wen, Francesca Luca, and Roger Pique-Regi. "Which Genetics Variants in DNase-Seq Footprints Are More Likely to Alter Binding?" PLOS Genetics 12, no. 2 (February 22, 2016): e1005875. http://dx.doi.org/10.1371/journal.pgen.1005875.

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Jankowski, Aleksander, Jerzy Tiuryn, and Shyam Prabhakar. "Romulus: robust multi-state identification of transcription factor binding sites from DNase-seq data." Bioinformatics 32, no. 16 (April 19, 2016): 2419–26. http://dx.doi.org/10.1093/bioinformatics/btw209.

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Mitra, Sneha, Jianling Zhong, Trung Q. Tran, David M. MacAlpine, and Alexander J. Hartemink. "RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data." Nucleic Acids Research 49, no. 14 (July 13, 2021): 7925–38. http://dx.doi.org/10.1093/nar/gkab553.

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Abstract Chromatin is a tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and occupancy levels of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. In contrast, epigenomic accessibility data like MNase-seq, DNase-seq, and ATAC-seq provide insight into the chromatin landscape of all factors bound along the genome, but with little insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin accessibility data with nucleotide sequence to jointly compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors. We apply RoboCOP to MNase-seq and ATAC-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome, and show that our model makes better predictions than existing methods. We also compute a chromatin occupancy profile of the yeast genome under cadmium stress, revealing chromatin dynamics associated with transcriptional regulation.

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He, Housheng Hansen, Clifford A. Meyer, Sheng'en Shawn Hu, Mei-Wei Chen, Chongzhi Zang, Yin Liu, Prakash K. Rao, et al. "Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification." Nature Methods 11, no. 1 (December 8, 2013): 73–78. http://dx.doi.org/10.1038/nmeth.2762.

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Chen, Ailing, Daozhen Chen, and Ying Chen. "Advances of DNase-seq for mapping active gene regulatory elements across the genome in animals." Gene 667 (August 2018): 83–94. http://dx.doi.org/10.1016/j.gene.2018.05.033.

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Frerichs, Anneke, Julia Engelhorn, Janine Altmüller, Jose Gutierrez-Marcos, and Wolfgang Werr. "Specific chromatin changes mark lateral organ founder cells in the Arabidopsis inflorescence meristem." Journal of Experimental Botany 70, no. 15 (April 30, 2019): 3867–79. http://dx.doi.org/10.1093/jxb/erz181.

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Abstract Fluorescence-activated cell sorting (FACS) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were combined to analyse the chromatin state of lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis apetala1-1 cauliflower-1 double mutant inflorescence meristem. On a genome-wide level, we observed a striking correlation between transposase hypersensitive sites (THSs) detected by ATAC-seq and DNase I hypersensitive sites (DHSs). The mostly expanded DHSs were often substructured into several individual THSs, which correlated with phylogenetically conserved DNA sequences or enhancer elements. Comparing chromatin accessibility with available RNA-seq data, THS change configuration was reflected by gene activation or repression and chromatin regions acquired or lost transposase accessibility in direct correlation with gene expression levels in LOFCs. This was most pronounced immediately upstream of the transcription start, where genome-wide THSs were abundant in a complementary pattern to established H3K4me3 activation or H3K27me3 repression marks. At this resolution, the combined application of FACS/ATAC-seq is widely applicable to detect chromatin changes during cell-type specification and facilitates the detection of regulatory elements in plant promoters.

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Savadel, Savannah D., Thomas Hartwig, Zachary M. Turpin, Daniel L. Vera, Pei-Yau Lung, Xin Sui, Max Blank, et al. "The native cistrome and sequence motif families of the maize ear." PLOS Genetics 17, no. 8 (August 12, 2021): e1009689. http://dx.doi.org/10.1371/journal.pgen.1009689.

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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.

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Chen, Jiaxin, Jian Zhang, Yu Gao, Yanyu Li, Chenchen Feng, Chao Song, Ziyu Ning, et al. "LncSEA: a platform for long non-coding RNA related sets and enrichment analysis." Nucleic Acids Research 49, no. D1 (October 12, 2020): D969—D980. http://dx.doi.org/10.1093/nar/gkaa806.

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Abstract Long non-coding RNAs (lncRNAs) have been proven to play important roles in transcriptional processes and various biological functions. Establishing a comprehensive collection of human lncRNA sets is urgent work at present. Using reference lncRNA sets, enrichment analyses will be useful for analyzing lncRNA lists of interest submitted by users. Therefore, we developed a human lncRNA sets database, called LncSEA, which aimed to document a large number of available resources for human lncRNA sets and provide annotation and enrichment analyses for lncRNAs. LncSEA supports >40 000 lncRNA reference sets across 18 categories and 66 sub-categories, and covers over 50 000 lncRNAs. We not only collected lncRNA sets based on downstream regulatory data sources, but also identified a large number of lncRNA sets regulated by upstream transcription factors (TFs) and DNA regulatory elements by integrating TF ChIP-seq, DNase-seq, ATAC-seq and H3K27ac ChIP-seq data. Importantly, LncSEA provides annotation and enrichment analyses of lncRNA sets associated with upstream regulators and downstream targets. In summary, LncSEA is a powerful platform that provides a variety of types of lncRNA sets for users, and supports lncRNA annotations and enrichment analyses. The LncSEA database is freely accessible at http://bio.liclab.net/LncSEA/index.php.

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Piper, Jason, Markus C. Elze, Pierre Cauchy, Peter N. Cockerill, Constanze Bonifer, and Sascha Ott. "Wellington: a novel method for the accurate identification of digital genomic footprints from DNase-seq data." Nucleic Acids Research 42, no. 17 (September 26, 2014): 11272. http://dx.doi.org/10.1093/nar/gku727.

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Piper, Jason, Markus C. Elze, Pierre Cauchy, Peter N. Cockerill, Constanze Bonifer, and Sascha Ott. "Wellington: a novel method for the accurate identification of digital genomic footprints from DNase-seq data." Nucleic Acids Research 41, no. 21 (September 25, 2013): e201-e201. http://dx.doi.org/10.1093/nar/gkt850.

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Giansanti, Valentina, Ming Tang, and Davide Cittaro. "Fast analysis of scATAC-seq data using a predefined set of genomic regions." F1000Research 9 (May 28, 2020): 199. http://dx.doi.org/10.12688/f1000research.22731.2.

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Background: Analysis of scATAC-seq data has been recently scaled to thousands of cells. While processing of other types of single cell data was boosted by the implementation of alignment-free techniques, pipelines available to process scATAC-seq data still require large computational resources. We propose here an approach based on pseudoalignment, which reduces the execution times and hardware needs at little cost for precision. Methods: Public data for 10k PBMC were downloaded from 10x Genomics web site. Reads were aligned to various references derived from DNase I Hypersensitive Sites (DHS) using kallisto and quantified with bustools. We compared our results with the ones publicly available derived by cellranger-atac. We subsequently tested our approach on scATAC-seq data for K562 cell line. Results: We found that kallisto does not introduce biases in quantification of known peaks; cells groups identified are consistent with the ones identified from standard method. We also found that cell identification is robust when analysis is performed using DHS-derived reference in place of de novo identification of ATAC peaks. Lastly, we found that our approach is suitable for reliable quantification of gene activity based on scATAC-seq signal, thus allows for efficient labelling of cell groups based on marker genes. Conclusions: Analysis of scATAC-seq data by means of kallisto produces results in line with standard pipelines while being considerably faster; using a set of known DHS sites as reference does not affect the ability to characterize the cell populations.

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Klein, David C., and Sarah J. Hainer. "Genomic methods in profiling DNA accessibility and factor localization." Chromosome Research 28, no. 1 (November 27, 2019): 69–85. http://dx.doi.org/10.1007/s10577-019-09619-9.

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AbstractRecent advancements in next-generation sequencing technologies and accompanying reductions in cost have led to an explosion of techniques to examine DNA accessibility and protein localization on chromatin genome-wide. Generally, accessible regions of chromatin are permissive for factor binding and are therefore hotspots for regulation of gene expression; conversely, genomic regions that are highly occupied by histone proteins are not permissive for factor binding and are less likely to be active regulatory regions. Identifying regions of differential accessibility can be useful to uncover putative gene regulatory regions, such as enhancers, promoters, and insulators. In addition, DNA-binding proteins, such as transcription factors that preferentially bind certain DNA sequences and histone proteins that form the core of the nucleosome, play essential roles in all DNA-templated processes. Determining the genomic localization of chromatin-bound proteins is therefore essential in determining functional roles, sequence motifs important for factor binding, and regulatory networks controlling gene expression. In this review, we discuss techniques for determining DNA accessibility and nucleosome positioning (DNase-seq, FAIRE-seq, MNase-seq, and ATAC-seq) and techniques for detecting and functionally characterizing chromatin-bound proteins (ChIP-seq, DamID, and CUT&RUN). These methods have been optimized to varying degrees of resolution, specificity, and ease of use. Here, we outline some advantages and disadvantages of these techniques, their general protocols, and a brief discussion of their development. Together, these complimentary approaches have provided an unparalleled view of chromatin architecture and functional gene regulation.

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Giansanti, Valentina, Ming Tang, and Davide Cittaro. "Fast analysis of scATAC-seq data using a predefined set of genomic regions." F1000Research 9 (March 20, 2020): 199. http://dx.doi.org/10.12688/f1000research.22731.1.

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Background: Analysis of scATAC-seq data has been recently scaled to thousands of cells. While processing of other types of single cell data was boosted by the implementation of alignment-free techniques, pipelines available to process scATAC-seq data still require large computational resources. We propose here an approach based on pseudoalignment, which reduces the execution times and hardware needs at little cost for precision. Methods: Public data for 10k PBMC were downloaded from 10x Genomics web site. Reads were aligned to various references derived from DNase I Hypersensitive Sites (DHS) using kallisto and quantified with bustools. We compared our results with the ones publicly available derived by cellranger-atac. Results: We found that kallisto does not introduce biases in quantification of known peaks and cells groups are identified in a consistent way. We also found that cell identification is robust when analysis is performed using DHS-derived reference in place of de novo identification of ATAC peaks. Lastly, we found that our approach is suitable for reliable quantification of gene activity based on scATAC-seq signal, thus allows for efficient labelling of cell groups based on marker genes. Conclusions: Analysis of scATAC-seq data by means of kallisto produces results in line with standard pipelines while being considerably faster; using a set of known DHS sites as reference does not affect the ability to characterize the cell populations

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Woo, Janghee, Sandra Stehling-Sun, H. Joachim Deeg, Thalia Papayannopoulou, Fyodor D. Urnov, and John A. Stamatoyannopoulos. "Regulatory Reprogramming of Erythropoiesis By DNMT3A Mutation." Blood 132, Supplement 1 (November 29, 2018): 4343. http://dx.doi.org/10.1182/blood-2018-99-120110.

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Abstract DNA methyltransferase 3A (DNMT3A) regulates diverse epigenetic processes, and DNMT3A mutations occur frequently in myelodysplastic syndromes (MDS), including in founding clones of MDS samples. Most DNMT3A mutations affect Arg882 (R882) in the catalytic domain of DNMT3A, and are found almost exclusively in a heterozygous state. To resolve the relationship between the genetic and epigenetic architectures of R882H+ MDS, we engineered primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to carry heterozygous DNMT3A R882H and performed temporally resolved, genome-wide regulatory mapping via DNase-seq combined with RNA-seq during erythroid differentiation in vitro, and in an in vivo transplantation model. Compared with isogenic controls, heterozygous R882H HSPCs cells exhibited markedly impaired erythroid differentiation, accumulation of early myeloid progenitors, and diverse maturation defects. Transplantation of R882H HSPCs into W41 NSG mice revealed both impaired erythroid differentiation and preferential survival of mutant alleles in multiple hematopoietic lineages compatible with an early progenitor defect. Regulatory profiling of DNMT3A R882H heterozygous cells during differentiation via combined DNase- and RNA-seq revealed global and sequential alterations in the regulatory landscapes in mutant cells, most prominently decommissioning of thousands of regulatory regions normally found in primitive cells that mark gene loci destined for expression during later differentiation stages. Decommissioned regulatory elements in R882H heterozygotes were concentrated around genes involved in both regulation of erythropoiesis and cell-cycle control, biasing HSPC differentiation away from erythropoiesis. Similar findings were observed in CD34+-selected bone marrows from 33 patients with MDS, comparing heterozygous DNMT3A R882H and wild type. Collectively, our results indicate that DNMT3A R882H mutation reprograms early myeloid regulatory landscapes by preferentially targeting elements that control genes destined to be expressed at later stages of differentiation, resulting in a combined phenotype of impaired myeloid differentiation, impaired erythroid maturation, and preferential survival of R882H+ cells. The results provide novel mechanistic insights into the chromatin programming of erythroid differentiation and its connection with MDS. Disclosures No relevant conflicts of interest to declare.

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Ho, Margaret C. W., Porfirio Quintero-Cadena, and Paul W. Sternberg. "Genome-wide discovery of active regulatory elements and transcription factor footprints in Caenorhabditis elegans using DNase-seq." Genome Research 27, no. 12 (October 26, 2017): 2108–19. http://dx.doi.org/10.1101/gr.223735.117.

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Degtyareva, Arina O., Elena V. Antontseva, and Tatiana I. Merkulova. "Regulatory SNPs: Altered Transcription Factor Binding Sites Implicated in Complex Traits and Diseases." International Journal of Molecular Sciences 22, no. 12 (June 16, 2021): 6454. http://dx.doi.org/10.3390/ijms22126454.

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The vast majority of the genetic variants (mainly SNPs) associated with various human traits and diseases map to a noncoding part of the genome and are enriched in its regulatory compartment, suggesting that many causal variants may affect gene expression. The leading mechanism of action of these SNPs consists in the alterations in the transcription factor binding via creation or disruption of transcription factor binding sites (TFBSs) or some change in the affinity of these regulatory proteins to their cognate sites. In this review, we first focus on the history of the discovery of regulatory SNPs (rSNPs) and systematized description of the existing methodical approaches to their study. Then, we brief the recent comprehensive examples of rSNPs studied from the discovery of the changes in the TFBS sequence as a result of a nucleotide substitution to identification of its effect on the target gene expression and, eventually, to phenotype. We also describe state-of-the-art genome-wide approaches to identification of regulatory variants, including both making molecular sense of genome-wide association studies (GWAS) and the alternative approaches the primary goal of which is to determine the functionality of genetic variants. Among these approaches, special attention is paid to expression quantitative trait loci (eQTLs) analysis and the search for allele-specific events in RNA-seq (ASE events) as well as in ChIP-seq, DNase-seq, and ATAC-seq (ASB events) data.

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Song, L., and G. E. Crawford. "DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Genome from Mammalian Cells." Cold Spring Harbor Protocols 2010, no. 2 (February 1, 2010): pdb.prot5384. http://dx.doi.org/10.1101/pdb.prot5384.

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Karamitros, Timokratis, Vasiliki Pogka, Gethsimani Papadopoulou, Ourania Tsitsilonis, Maria Evangelidou, Styliani Sympardi, and Andreas Mentis. "Dual RNA-Seq Enables Full-Genome Assembly of Measles Virus and Characterization of Host–Pathogen Interactions." Microorganisms 9, no. 7 (July 20, 2021): 1538. http://dx.doi.org/10.3390/microorganisms9071538.

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Measles virus (MeV) has a negative-sense 15 kb long RNA genome, which is generally conserved. Recent advances in high-throughput sequencing (HTS) and Dual RNA-seq allow the analysis of viral RNA genomes and the discovery of viral infection biomarkers, via the simultaneous characterization of the host transcriptome. However, these host–pathogen interactions remain largely unexplored in MeV infections. We performed untargeted Dual RNA-seq in 6 pharyngeal and 6 peripheral blood mononuclear cell (PBMCs) specimens from patients with MeV infection, as confirmed via routine real-time PCR testing. Following optimised DNase treatment of total nucleic acids, we used the pharyngeal samples to build poly-A-enriched NGS libraries. We reconstructed the viral genomes using the pharyngeal datasets and we further conducted differential expression, gene-ontology and pathways enrichment analysis to compare both the pharyngeal and the peripheral blood transcriptomes of the MeV-infected patients vs. control groups of healthy individuals. We obtained 6 MeV genotype-B3 full-genome sequences. We minutely analyzed the transcriptome of the MeV-infected pharyngeal epithelium, detecting all known viral infection biomarkers, but also revealing a functional cluster of local antiviral and inflammatory immune responses, which differ substantially from those observed in the PBMCs transcriptome. The application of Dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the virus genome structure and the cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

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Inoue, Kazuki, and Yuuki Imai. "Identification of Novel Transcription Factors in Osteoclast Differentiation Using Genome-wide Analysis of Open Chromatin Determined by DNase-seq." Journal of Bone and Mineral Research 29, no. 8 (July 21, 2014): 1823–32. http://dx.doi.org/10.1002/jbmr.2229.

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Behera, Vivek, Perry Evans, Carolyne J. Face, Laavanya Sankaranarayanan, and Gerd A. Blobel. "Deep Mining of Natural Genetic Variation in Erythroid Cells Reveals New Insights about In Vivo Transcription Factor Binding and Chromatin Accessibility." Blood 128, no. 22 (December 2, 2016): 3879. http://dx.doi.org/10.1182/blood.v128.22.3879.3879.

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Abstract Erythroid transcription factors (TFs) control gene expression programs, lineage decisions, and disease outcomes. How transcription factors contact DNA has been studied extensively in vitro, but in vivo binding characteristics are less well understood as they are influenced in a reciprocal manner by chromatin accessibility and neighboring transcription factors. Here, we present a comparative analysis approach that takes advantage of non-coding sequence variation between functionally equivalent erythroid cell lines to conduct an in-depth analysis of erythroid TF binding profiles and chromatin features. Specifically, we analyzed ChIP-seq datasets to identify millions of genetic non-coding variants between the mouse erythroleukemia cell line (MEL), a GATA1-inducible erythroid progenitor cell line (G1E-ER4), and primary murine erythroblast cells. We found that while these cell lines are highly positively correlated in chromatin features, larger differences in TF binding intensity are correlated with higher degrees of genetic variation between cell lines. We next examined discriminatory genetic variants between the cell lines that are located in ChIP-seq peaks of the erythroid transcription factor GATA1. Hundreds of such variants fall within GATA1 motifs. Differential GATA1 binding intensities associated with the variants revealed nucleotide positions that contribute most to in vivo GATA1 chromatin occupancy and identified which alternative nucleotides are most likely to disrupt binding. Notably, this additional information about GATA1's in vivo nucleotide binding preferences improved prediction of GATA1 binding sites genome-wide. We applied similar approaches to determine the bp-resolution in vivo binding preferences of TAL1/SCL and CTCF. We additionally identified thousands of discriminatory genetic variants within GATA1 sites that fall outside canonical GATA elements but within binding sites of other known TFs. Association of these variants with differential GATA1 binding intensities revealed that the hematopoietic transcription factors TAL1/SCL and KLF1 positively regulate GATA1 chromatin occupancy. Strikingly, we identified a number of motifs not previously implicated in cooperating with GATA1 that positively impact GATA1 chromatin binding. Notably, we also defined motifs associated with negative regulation of GATA1 chromatin occupancy. Applying a similar analysis to TAL1/SCL and CTCF revealed additional motifs involved in regulating the chromatin occupancy of these TFs. Finally, we associated discriminatory genetic variation between erythroid cell lines with large changes in sub-kb-scale DNase hypersensitivity. We found that single base pair substitutions within or near a number of erythroid TF motifs, including that for the RUNX family of nuclear factors, are strongly associated with changes in chromatin accessibility. Our findings use novel methods in comparative ChIP-seq and DNase-seq analysis to reveal new insights about the genetic basis for erythroid TF chromatin occupancy and chromatin accessibility. Disclosures No relevant conflicts of interest to declare.

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Kianmehr, Khazali, Rajabi-Maham, Sharifi-Zarchi, Cuzin, and Rassoulzadegan. "Genome-Wide Distribution of Nascent Transcripts in Sperm DNA, Products of a Late Wave of General Transcription." Cells 8, no. 10 (October 3, 2019): 1196. http://dx.doi.org/10.3390/cells8101196.

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Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

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Raxwal, Vivek Kumar, Sourav Ghosh, Somya Singh, Surekha Katiyar-Agarwal, Shailendra Goel, Arun Jagannath, Amar Kumar, Vinod Scaria, and Manu Agarwal. "Abiotic stress-mediated modulation of the chromatin landscape in Arabidopsis thaliana." Journal of Experimental Botany 71, no. 17 (June 11, 2020): 5280–93. http://dx.doi.org/10.1093/jxb/eraa286.

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Abstract Limited information is available on abiotic stress-mediated alterations of chromatin conformation influencing gene expression in plants. In order to characterize the effect of abiotic stresses on changes in chromatin conformation, we employed FAIRE-seq (formaldehyde-assisted isolation of regulatory element sequencing) and DNase-seq to isolate accessible regions of chromatin from Arabidopsis thaliana seedlings exposed to either heat, cold, salt, or drought stress. Approximately 25% of regions in the Arabidopsis genome were captured as open chromatin, the majority of which included promoters and exons. A large proportion of chromatin regions apparently did not change their conformation in response to any of the four stresses. Digital footprints present within these regions had differential enrichment of motifs for binding of 43 different transcription factors. Further, in contrast to drought and salt stress, both high and low temperature treatments resulted in increased accessibility of the chromatin. Also, pseudogenes attained increased chromatin accessibility in response to cold and drought stresses. The highly accessible and inaccessible chromatin regions of seedlings exposed to drought stress correlated with the Ser/Thr protein kinases (MLK1 and MLK2)-mediated reduction and increase in H3 phosphorylation (H3T3Ph), respectively. The presented results provide a deeper understanding of abiotic stress-mediated chromatin modulation in plants.

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Jaroszewicz, Artur, and Jason Ernst. "An integrative approach for fine-mapping chromatin interactions." Bioinformatics 36, no. 6 (November 19, 2019): 1704–11. http://dx.doi.org/10.1093/bioinformatics/btz843.

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Abstract Motivation Chromatin interactions play an important role in genome architecture and gene regulation. The Hi-C assay generates such interactions maps genome-wide, but at relatively low resolutions (e.g. 5-25 kb), which is substantially coarser than the resolution of transcription factor binding sites or open chromatin sites that are potential sources of such interactions. Results To predict the sources of Hi-C-identified interactions at a high resolution (e.g. 100 bp), we developed a computational method that integrates data from DNase-seq and ChIP-seq of TFs and histone marks. Our method, χ-CNN, uses this data to first train a convolutional neural network (CNN) to discriminate between called Hi-C interactions and non-interactions. χ-CNN then predicts the high-resolution source of each Hi-C interaction using a feature attribution method. We show these predictions recover original Hi-C peaks after extending them to be coarser. We also show χ-CNN predictions enrich for evolutionarily conserved bases, eQTLs and CTCF motifs, supporting their biological significance. χ-CNN provides an approach for analyzing important aspects of genome architecture and gene regulation at a higher resolution than previously possible. Availability and implementation χ-CNN software is available on GitHub (https://github.com/ernstlab/X-CNN). Supplementary information Supplementary data are available at Bioinformatics online.

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Zhang, Jun, Bing Liu, Dan Gu, Yuan Hao, Mo Chen, Yue Ma, Xiaohui Zhou, David Reverter, Yuanxing Zhang, and Qiyao Wang. "Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression." Nucleic Acids Research 49, no. 6 (March 8, 2021): 3274–93. http://dx.doi.org/10.1093/nar/gkab150.

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Abstract LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.

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Chumsakul, O., K. Nakamura, T. Kurata, T. Sakamoto, J. L. Hobman, N. Ogasawara, T. Oshima, and S. Ishikawa. "High-Resolution Mapping of In vivo Genomic Transcription Factor Binding Sites Using In situ DNase I Footprinting and ChIP-seq." DNA Research 20, no. 4 (April 11, 2013): 325–38. http://dx.doi.org/10.1093/dnares/dst013.

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Pilon, Andre M., Elliott H. Margulies, Hatice Ozel Abaan, Amy Werner Allen, Tim M. Townes, Abbie M. Frederick, Dewang Zhou, Patrick G. Gallagher, and David M. Bodine. "Genome-Wide Analysis of EKLF Occupancy in Erythroid Chromatin Reveals 5′, 3′ and Intragenic Binding Sites in EKLF Target Genes." Blood 112, no. 11 (November 16, 2008): 283. http://dx.doi.org/10.1182/blood.v112.11.283.283.

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Abstract Erythroid Kruppel-Like Factor (EKLF; KLF1) is the founding member of the Kruppel family of transcription factors, with 3 C2H2 zinc-fingers that bind a 9-base consensus sequence (NCNCNCCCN). The functions of EKLF, first identified as an activator of the beta-globin locus, include gene activation and chromatin remodeling. Our knowledge of genes regulated by EKLF is limited, as EKLF-deficient mice die by embryonic day 15 (E15), due to a severe anemia. Analysis of E13.5 wild type and EKLF-deficient fetal liver (FL) erythroid cells revealed that EKLF-deficient cells fail to complete terminal erythroid maturation (Pilon et al. submitted). Coupling chromatin immunoprecipitation and ultra high-throughput massively parallel sequencing (ChIP-seq) is increasingly being used for mapping protein-DNA interactions in vivo on a genome-wide scale. ChIP-seq allows a simultaneous analysis of transcription factor binding in every region of the genome, defining an “interactome”. To elucidate direct EKLF-dependent effects on erythropoiesis, we have combined ChIP-seq with expression array (“transcriptome”) analyses. We feel that integration of ChIP-seq and microarray data can provide us detailed knowledge of the role of EKLF in erythropoiesis. Chromatin was isolated from E13.5 FL cells of mice whose endogenous EKLF gene was replaced with a fully functional HA-tagged EKLF gene. ChIP was performed using a highly specific high affinity anti-HA antibody. A library of EKLF-bound FL chromatin enriched by anti-HA IP was created and subjected to fluorescent in situ sequencing on a Solexa 1G platform, providing 36-base signatures that were mapped to unique sites in the mouse genome, defining the EKLF “interactome.” The frequency with which a given signature appears provides a measurable peak of enrichment. We performed three biological/technical replicates and analyzed each data set individually as well as the combined data. To validate ChIP-seq results, we examined the locus of a known EKLF target gene, a-hemoglobin stabilizing protein (AHSP). Peaks corresponded to previously identified DNase hypersensitive sites, regions of histone hyperacetylation, and sites of promoter-occupancy determined by ChIP-PCR. A genome wide analysis, focusing on the regions with the highest EKLF occupancy revealed a set of 531 locations where high levels EKLF binding occurs. Of these sites, 119 (22%) are located 10 kb or more from the nearest gene and are classified as intergenic EKLF binding sites. Another 78 sites (14.6%) are within 10 kb of an annotated RefSeq gene. A plurality of the binding sites, 222 (42%), are within RefSeq coordinates and are classified as intragenic EKLF binding sites. Microarray profiling of mRNA from sorted, matched populations of dE13.5 WT and EKLF-deficient FL erythroid progenitor cells showed dysregulation of >3000 genes (p<0.05). Ingenuity Pathways Analysis (IPA) of the >3000 dysregulated mRNAs indicated significant alteration of a cell cycle-control network, centered about the transcription factor, E2f2. We confirmed significantly decreased E2f2 mRNA and protein levels by real-time PCR and Western blot, respectively; demonstrated that EKLF-deficient FL cells accumulate in G0/G1 by cell cycle analysis; and verified EKLF-binding to motifs within the E2f2 promoter by ChIP-PCR and analysis of the ChIP Seq data. We hypothesized that only a subset of the 3000 dysregulated genes would be direct EKLF targets. We limited the ChIP-seq library to display the top 5% most frequently represented fragments across the genome, and applied this criterion to the network of dysregulated mRNAs in the IPA cell cycle network. ChIP-seq identified peaks of EKLF association with 60% of the loci in this pathway. However, consistent with the role of EKLF as a transcriptional activator, 95% of the occupied genomic loci corresponded to mRNAs whose expression in EKLF-deficient FL cells was significantly decreased (p<0.05). The majority (59%) of these EKLF-bound sites were located at intragenic sites (i.e., introns), while a minority (15% and 26%) were found adjacent to the genes or in intergenic regions. We have shown that both the AHSP and E2f2 loci require EKLF to cause the locus to become activated and sensitive to DNase I digestion in erythroid cells. Based on the increased frequency of intragenic EKLF-binding sites, particularly in genes of the cell cycle network, we propose that the occupancy of intragenic sites by EKLF may facilitate chromatin modification.

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Martynova, Elena, Yilin Zhao, Qing Xie, Deyou Zheng, and Ales Cvekl. "Transcriptomic analysis and novel insights into lens fibre cell differentiation regulated by Gata3." Open Biology 9, no. 12 (December 2019): 190220. http://dx.doi.org/10.1098/rsob.190220.

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Gata3 is a DNA-binding transcription factor involved in cellular differentiation in a variety of tissues including inner ear, hair follicle, kidney, mammary gland and T-cells. In a previous study in 2009, Maeda et al . ( Dev. Dyn. 238 , 2280–2291; doi:10.1002/dvdy.22035 ) found that Gata3 mutants could be rescued from midgestational lethality by the expression of a Gata3 transgene in sympathoadrenal neuroendocrine cells. The rescued embryos clearly showed multiple defects in lens fibre cell differentiation. To determine whether these defects were truly due to the loss of Gata3 expression in the lens, we generated a lens-specific Gata3 loss-of-function model. Analogous to the previous findings, our Gata3 null embryos showed abnormal regulation of cell cycle exit during lens fibre cell differentiation, marked by reduction in the expression of the cyclin-dependent kinase inhibitors Cdkn1b/p27 and Cdkn1c/p57, and the retention of nuclei accompanied by downregulation of Dnase IIβ. Comparisons of transcriptomes between control and mutated lenses by RNA-Seq revealed dysregulation of lens-specific crystallin genes and intermediate filament protein Bfsp2. Both Cdkn1b/p27 and Cdkn1c/p57 loci are occupied in vivo by Gata3, as well as Prox1 and c-Jun, in lens chromatin. Collectively, our studies suggest that Gata3 regulates lens differentiation through the direct regulation of the Cdkn1b/p27and Cdkn1c/p57 expression, and the direct/or indirect transcriptional control of Bfsp2 and Dnase IIβ.

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Ferreira, Leonardo M. R., Torsten B. Meissner, Tarjei S. Mikkelsen, William Mallard, Charles W. O’Donnell, Tamara Tilburgs, Hannah A. B. Gomes, et al. "A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface." Proceedings of the National Academy of Sciences 113, no. 19 (April 13, 2016): 5364–69. http://dx.doi.org/10.1073/pnas.1602886113.

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HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.

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Sieber, Karsten B., Anna Batorsky, Kyle Siebenthall, Kelly L. Hudkins, Jeff D. Vierstra, Shawn Sullivan, Aakash Sur, et al. "Integrated Functional Genomic Analysis Enables Annotation of Kidney Genome-Wide Association Study Loci." Journal of the American Society of Nephrology 30, no. 3 (February 13, 2019): 421–41. http://dx.doi.org/10.1681/asn.2018030309.

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BackgroundLinking genetic risk loci identified by genome-wide association studies (GWAS) to their causal genes remains a major challenge. Disease-associated genetic variants are concentrated in regions containing regulatory DNA elements, such as promoters and enhancers. Although researchers have previously published DNA maps of these regulatory regions for kidney tubule cells and glomerular endothelial cells, maps for podocytes and mesangial cells have not been available.MethodsWe generated regulatory DNA maps (DNase-seq) and paired gene expression profiles (RNA-seq) from primary outgrowth cultures of human glomeruli that were composed mainly of podocytes and mesangial cells. We generated similar datasets from renal cortex cultures, to compare with those of the glomerular cultures. Because regulatory DNA elements can act on target genes across large genomic distances, we also generated a chromatin conformation map from freshly isolated human glomeruli.ResultsWe identified thousands of unique regulatory DNA elements, many located close to transcription factor genes, which the glomerular and cortex samples expressed at different levels. We found that genetic variants associated with kidney diseases (GWAS) and kidney expression quantitative trait loci were enriched in regulatory DNA regions. By combining GWAS, epigenomic, and chromatin conformation data, we functionally annotated 46 kidney disease genes.ConclusionsWe demonstrate a powerful approach to functionally connect kidney disease-/trait–associated loci to their target genes by leveraging unique regulatory DNA maps and integrated epigenomic and genetic analysis. This process can be applied to other kidney cell types and will enhance our understanding of genome regulation and its effects on gene expression in kidney disease.

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Liu, Jialin, Rebecca R. Viales, Pierre Khoueiry, James P. Reddington, Charles Girardot, Eileen E. M. Furlong, and Marc Robinson-Rechavi. "The hourglass model of evolutionary conservation during embryogenesis extends to developmental enhancers with signatures of positive selection." Genome Research 31, no. 9 (July 15, 2021): 1573–81. http://dx.doi.org/10.1101/gr.275212.121.

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Inter-species comparisons of both morphology and gene expression within a phylum have revealed a period in the middle of embryogenesis with more similarity between species compared with earlier and later time points. This “developmental hourglass” pattern has been observed in many phyla, yet the evolutionary constraints on gene expression, as well as the underlying mechanisms of how this is regulated, remain elusive. Moreover, the role of positive selection on gene regulation in the more diverged earlier and later stages of embryogenesis remains unknown. Here, using DNase-seq to identify regulatory regions in two distant Drosophila species (D. melanogaster and D. virilis), we assessed the evolutionary conservation and adaptive evolution of enhancers throughout multiple stages of embryogenesis. This revealed a higher proportion of conserved enhancers at the phylotypic period, providing a regulatory basis for the hourglass expression pattern. Using an in silico mutagenesis approach, we detect signatures of positive selection on developmental enhancers at early and late stages of embryogenesis, with a depletion at the phylotypic period, suggesting positive selection as one evolutionary mechanism underlying the hourglass pattern of animal evolution.

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Shyamsunder, Pavithra, Mahalakshmi Shanmugasundaram, Anand Mayakonda, Weoi Woon Teoh, Lin Han, Mei Chee Lim, Melissa Fullwood, et al. "Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation." Blood 132, Supplement 1 (November 29, 2018): 3834. http://dx.doi.org/10.1182/blood-2018-99-116091.

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Abstract CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins. Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE. Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp). To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice. In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

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Kean, Thomas, Zhongqi Ge, Yumei Li, Rui Chen, and James Dennis. "Transcriptome-Wide Analysis of Human Chondrocyte Expansion on Synoviocyte Matrix." Cells 8, no. 2 (January 24, 2019): 85. http://dx.doi.org/10.3390/cells8020085.

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Human chondrocytes are expanded and used in autologous chondrocyte implantation techniques and are known to rapidly de-differentiate in culture. These chondrocytes, when cultured on tissue culture plastic (TCP), undergo both phenotypical and morphological changes and quickly lose the ability to re-differentiate to produce hyaline-like matrix. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation, allowing for more than twice the number of population doublings (PD) whilst retaining chondrogenic capacity. The goal of this study was to apply RNA sequencing (RNA-Seq) analysis to examine the differences between TCP-expanded and SDECM-expanded human chondrocytes. Human chondrocytes from three donors were thawed from primary stocks and cultured on TCP flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. During log expansion, RNA was extracted from the cell layer (70–90% confluence) at passages 1 and 4. Total RNA was column-purified and DNAse-treated before quality control analysis and next-generation RNA sequencing. Significant effects on gene expression were observed due to both culture surface and passage number. These results offer insight into the mechanism of how SDECM provides a more chondrogenesis-preserving environment for cell expansion, the transcriptome-wide changes that occur with culture, and potential mechanisms for further enhancement of chondrogenesis-preserving growth.

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Martínez-García, Pedro Manuel, Miguel García-Torres, Federico Divina, José Terrón-Bautista, Irene Delgado-Sainz, Francisco Gómez-Vela, and Felipe Cortés-Ledesma. "Genome-wide prediction of topoisomerase IIβ binding by architectural factors and chromatin accessibility." PLOS Computational Biology 17, no. 1 (January 19, 2021): e1007814. http://dx.doi.org/10.1371/journal.pcbi.1007814.

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DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.

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Krsteski, Jovan, Mario Gorenjak, Igor But, Maja Pakiž, and Uroš Potočnik. "Dysregulation of Synaptic Signaling Genes Is Involved in Biology of Uterine Leiomyoma." Genes 12, no. 8 (July 29, 2021): 1179. http://dx.doi.org/10.3390/genes12081179.

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Uterine leiomyomas are tumors, which are hormone driven and originate from the smooth muscle layer of the uterine wall. In addition to known genes in leiomyoma pathogenesis, recent approaches also highlight epigenetic malfunctions as an important mechanism of gene dysregulation. RNA sequencing raw data from pair-matched normal myometrium and fibroid tumors from two independent studies were used as discovery and validation sets and reanalyzed. RNA extracted from normal myometrium and fibroid tumors from 58 Slovenian patients was used as independent confirmation of most significant differentially expressed genes. Subsequently, GWA data from leiomyoma patients were used in order to identify genetic variants at epigenetic marks. Gene Ontology analysis of the overlap of two independent RNA-seq analyses showed that NPTX1, NPTX2, CHRM2, DRD2 and CACNA1A were listed as significant for several enriched GO terms. All five genes were subsequently confirmed in the independent Slovenian cohort. Additional integration and functional analysis showed that genetic variants in these five gene regions are listed at a chromatin structure and state, predicting promoters, enhancers, DNase hypersensitivity and altered transcription factor binding sites. We identified a unique subgroup of dysregulated synaptic signaling genes involved in the biology and pathogenesis of leiomyomas, adding to the complexity of tumor biology.

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Fang, Xiangdong, Kai-Hsin Chang, Daniel Bates, Morgan Diegel, Richard Sandstrom, Molly Weaver, Michael O. Dorschner, et al. "Chromatin Profiling of the Globin Loci of Human ES Cells and ES-Derived Erythroid Cells." Blood 114, no. 22 (November 20, 2009): 2535. http://dx.doi.org/10.1182/blood.v114.22.2535.2535.

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Abstract Abstract 2535 Poster Board II-512 We used a high throughput approach to determine the chromatin profiles of the human β and α globin loci and their upstream and downstream regions in human undifferentiated ES cells, ES cell-derived erythroid cells, human fetal and adult origin erythroid cells and in primary cells and cell lines of endo-meso and ectodermal origins. All DNase I hypersensitive sites of the b-locus were absent in undifferentiated human ES cells except for HS2 of the b-globin locus control region. The chromatin profiles of the β and α globin loci of ES cell-derived erythroid cells were identical to those of fetal liver erythroid cells except that the hypersensitive site of the embryonic globin gene was more prominent. DNase I hypersensitive site 2 of the b-globin LCR, a potent enhancer, was present in all the cell lines and primary lineages we studied, providing direct evidence that it is ubiquitous. Several new erythroid specific DHSs were detected upstream of 5′HS7 of the β-LCR, raising the possibility that they play a role in the regulation of the β globin locus. The region downstream to 3′HS1 was depleted of DHSs except for the previously identified DHS mapping near the breakpoint of HPFH 1. Since DHSs are absent near the breakpoints of deletional HPFHs and db thalassemias and since enhancers are typically DHS positive, our results argue against the hypothesis of imported enhancers in the pathogenesis of deletional HPFH and db thalassemia mutants. All the previously identified erythroid specific DHSs of the α globin locus were absent in human ES cells. The α globin locus of ES cells, however, displayed three very prominent DHSs, which were located almost symmetrically about 40 Kb apart from each other and they were constitutively formed in all the lineages and cell lines we have studied; the 3′ and 5′ DHSs carried CTCF sites by ChIP-Seq assay raising the possibility that they mark the sites of chromatin insulators. Overall these results demonstrate the power of the new high throughput chromatin profiling approaches and their ability to uncover features of chromatin that may be of regulatory relevance. Disclosures: No relevant conflicts of interest to declare.

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Journal articles on the topic 'DNase-seq'

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