Impact of the gut microbiota on enhancer accessibility in gut intraepithelial lymphocytes

The gut microbiota impacts many aspects of host biology including immune function. One hypothesis is that microbial communities induce epigenetic changes with accompanying alterations in chromatin accessibility, providing a mechanism that allows a community to have sustained host effects even in the face of its structural or functional variation. We used Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to define chromatin accessibility in predicted enhancer regions of intestinal αβ+and γδ+intraepithelial lymphocytes purified from germ-free mice, their conventionally raised (CONV-R) counterparts, and mice reared germ free and then colonized with CONV-R gut microbiota at the end of the suckling–weaning transition. Characterizing genes adjacent to traditional enhancers and super-enhancers revealed signaling networks, metabolic pathways, and enhancer-associated transcription factors affected by the microbiota. Our results support the notion that epigenetic modifications help define microbial community-affiliated functional features of host immune cell lineages.

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ATAC-seq with unique molecular identifiers improves quantification and footprinting

Communications Biology ◽

10.1038/s42003-020-01403-4 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Tao Zhu ◽

Keyan Liao ◽

Rongfang Zhou ◽

Chunjiao Xia ◽

Weibo Xie

Keyword(s):

Transcription Factor ◽

High Throughput ◽

High Throughput Sequencing ◽

Pcr Amplification ◽

Chromatin Accessibility ◽

Link Type ◽

Pcr Duplicates ◽

Identify Transcription Factor ◽

Accessible Chromatin ◽

Accurate Quantification

AbstractATAC-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.

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Development of early-stage type 1 diabetes in germ-free interleukin-10 deficient mice

10.1101/2020.04.18.048272 ◽

2020 ◽

Author(s):

Alexandria M. Bobe ◽

Jun Miyoshi ◽

Patrick Moore ◽

Suzanne Devkota ◽

Vanessa Leone ◽

...

Keyword(s):

Type 1 Diabetes ◽

Gut Microbiota ◽

Immune Cell ◽

Early Stage ◽

Myeloid Cell ◽

Interleukin 10 ◽

Experimental Models ◽

Germ Free ◽

Early Stages

AbstractSeveral experimental models demonstrate a role for gut microbiota in the progression of type 1 diabetes (T1D) in genetically prone hosts. While the association between disturbances in gut microbiota, or microbial dysbiosis, and complex immune diseases such as inflammatory bowel diseases (IBD) are well established, less is known about its role in T1D pathogenesis. In IBD-prone interleukin-10 deficient (IL-10 KO) mice, the absence of gut microbiota under germ-free (GF) conditions prevents IBD development. However, in aged GF IL-10 KO mice (>6-months of age), polyuria and pancreatic lymphocytic infiltration resembling T1D lesions was observed. Approximately 50% of male and female mice above 6-months of age develop pancreatic immune cell infiltration, as compared to none in conventionally-raised and fecal microbiota transplanted (FMT) IL-10 KO counterparts. Immunofluorescence staining of islet infiltrates was positive for adaptive and innate immunological markers, including lymphoid and myeloid cell markers, which typically characterize autoimmune T1D lesions. A subset of GF IL-10 KO mice was also positive for insulin autoantibodies (IAA), but the majority of mice did not become diabetic. Our findings of early stage lymphocytic infiltrates in the pancreas and IAA in the absence of overt diabetes in GF IL-10 KO mice embody the early stages of T1D pathogenesis. As such, we propose that the presence of gut microbiota play a protective role against immune infiltration in the pancreas of genetically prone hosts. Moreover, our model provides an opportunity to better understand the role of the microbiota in the early stages of immune pathogenesis and perhaps conceive the development of microbe-mediated prophylactic strategies to treat or even prevent T1D.

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Dendritic cell CX3CR1 and macrophages F4/80 play a central role in between gut micro biome and inflammation in Arsenic induced mice

10.1101/2021.01.25.428065 ◽

2021 ◽

Author(s):

Chiranjeevi Tikka ◽

Ram Kumar Manthari ◽

Ruiyan Niu ◽

Zilong Sun ◽

Jundong Wang

Keyword(s):

Immune System ◽

Gut Microbiota ◽

Chronic Exposure ◽

High Throughput Sequencing ◽

Immune Cell ◽

Underlying Mechanism ◽

Sequencing Analysis ◽

Protein Levels ◽

Transmission Electron ◽

Intestinal Immune System

AbstractMicrobiota plays a crucial role to protect the intestine contrary to the harmful foreign microorganisms and organize the immune system via numerous mechanisms, which include either direct or indirect environmental factors. The underlying mechanism arsenic (As) influenced immune system and regulates inflammation by altering gut microbiome in ileum remains unclear. However, chronic exposure to arsenic (at doses of 0.15 mg or 1.5 mg or 15 mg As2O3/ L in drinking water) significantly increased mRNA and protein levels of F4/80 and CX3CR1, concurrently, the increased levels of mRNA and protein IFNγ, TNFα, IL-18 and decreased levels of IL-10 were found in both 3 and 6 months exposure periods. High-throughput sequencing analysis revealed that gut microbiota at phylum; family and taxonomical levels were showed the abundance of gut microbiota. Evidentially, the ultra-structure of intestinal villi, microbes engulfed and immune cell migration were showed by the transmission electron microscopy. Chronic exposure to As influenced the inflammation by changing immune system and altered gut microbiota. In this study we conclude that chronic exposure to As breakdown the normal gut microbial community and increase the pathogenicity, the resultant risk pathogen direct contact with intestinal immune system and regulate the inflammation.

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Differential chromatin accessibility landscape reveals the structural and functional features of the allopolyploid wheat chromosomes

10.1101/2020.05.04.076737 ◽

2020 ◽

Cited By ~ 1

Author(s):

Katherine W. Jordan ◽

Fei He ◽

Monica Fernandez de Soto ◽

Alina Akhunova ◽

Eduard Akhunov

Keyword(s):

Agronomic Traits ◽

Chromatin Accessibility ◽

Genomic Variation ◽

Wheat Genome ◽

Gene Encoding ◽

Chromatin States ◽

Chromosomal Position ◽

Intergenic Regions ◽

Functional Features ◽

Accessible Chromatin

AbstractBackgroundWe have a limited understanding of how the complexity of the wheat genome influences the distribution of chromatin states along the homoeologous chromosomes. Using a differential nuclease sensitivity (DNS) assay, we investigated the chromatin states in the coding and transposon element (TE) -rich repetitive regions of the allopolyploid wheat genome.ResultsWe observed a negative chromatin accessibility gradient along the telomere-centromere axis with mostly open and closed chromatin located in the distal and pericentromeric regions of chromosomes, respectively. This trend was mirrored by the TE-rich intergenic regions, but not by the genic regions, which showed similar averages of chromatin accessibility levels along the chromosomes. The genes’ proximity to TEs was negatively associated with chromatin accessibility. The chromatin states of TEs was dependent on their type, proximity to genes, and chromosomal position. Both the distance between genes and TE composition appear to play a more important role in the chromatin accessibility along the chromosomes than chromosomal position. The majority of MNase hypersensitive regions were located within the TEs. The DNS assay accurately predicted previously detected centromere locations. SNPs located within more accessible chromatin explain a higher proportion of genetic variance for a number of agronomic traits than SNPs located within closed chromatin.ConclusionsThe chromatin states in the wheat genome are shaped by the interplay of repetitive and gene-encoding regions that are predictive of the functional and structural organization of chromosomes, providing a powerful framework for detecting genomic features involved in gene regulation and prioritizing genomic variation to explain phenotypes.

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Deep characterization of paired chromatin and transcriptomes in four immune cell types from multiple sclerosis patients

Epigenomics ◽

10.2217/epi-2021-0205 ◽

2021 ◽

Author(s):

Sunjay Jude Fernandes ◽

Matilda Ericsson ◽

Mohsen Khademi ◽

Maja Jagodic ◽

Tomas Olsson ◽

...

Keyword(s):

Multiple Sclerosis ◽

Immune Cell ◽

Cell Types ◽

Chromatin Accessibility ◽

Primary Role ◽

Nucleotide Polymorphisms ◽

Multiple Sclerosis Patients ◽

Accessible Chromatin

Background: The putative involvement of chromatin states in multiple sclerosis (MS) is thus far unclear. Here we determined the association of chromatin-accessibility with concurrent genetic, epigenetic and transcriptional events. Material & methods: We generated paired assay for transposase-accessible chromatin sequencing and RNA-seq profiles from sorted blood immune CD4+ and CD8+ T cells, CD14+ monocytes and CD19+ B cells from healthy controls (HCs) and MS patients. Results: We identified differentially accessible regions between MS and HCs, primarily in CD4+ and CD19+. CD4+ regions were enriched for MS-associated single nucleotide polymorphisms and differentially methylated loci. In the vicinity of differentially accessible regions of CD4+ cells, 42 differentially expressed genes were identified. The top two dysregulated genes identified in this multilayer analysis were CCDC114 and SERTAD1. Conclusion: These findings provide new insight into the primary role of CD4+ and CD19+ cells in MS.

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Successful ATAC-Seq From Snap-Frozen Equine Tissues

Frontiers in Genetics ◽

10.3389/fgene.2021.641788 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sichong Peng ◽

Rebecca Bellone ◽

Jessica L. Petersen ◽

Theodore S. Kalbfleisch ◽

Carrie J. Finno

Keyword(s):

Functional Annotation ◽

High Throughput Sequencing ◽

Chromatin Accessibility ◽

Tissue Type ◽

Tissue Samples ◽

Genome Wide ◽

Frozen Tissue ◽

And Storage ◽

Accessible Chromatin ◽

Animal Genomes

An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.

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Assessing chromatin accessibility in maize using ATAC-seq

10.1101/526079 ◽

2019 ◽

Author(s):

Yi-Jing Lee ◽

Pearl Chang ◽

Jui-Hsien Lu ◽

Pao-Yang Chen ◽

Chung-Ju Rachel Wang

Keyword(s):

Cell Sorting ◽

High Throughput Sequencing ◽

Crop Improvement ◽

Practical Method ◽

Chromatin Accessibility ◽

Crop Species ◽

Chromatin Profiling ◽

Key Steps ◽

Accessible Chromatin ◽

Complex Genome

Background: Maize is an important crop that has a complex genome. A better understanding of maize chromatin architecture provides great opportunities for crop improvement, because chromatin accessibility influences gene expression, thereby affecting agricultural traits. The newly developed method for chromatin profiling, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), has been developed to investigate chromatin accessibility. Result: We adapt this method by testing parameters of several key steps and generate the first ATAC-seq protocol for maize. We demonstrate that purification of maize nuclei to eliminate organelles can be achieved without the need for cell sorting, and that only a standard bench-top centrifuge is required for sample preparation. Finally, our sequence analyses confirm that our protocol of ATAC-seq can be successfully used to assess the chromatin landscape in maize. Conclusion: The ATAC-seq provides a useful technique to study the chromatin accessibility. Given the parameters tested in our study, it can be a simple and practical method for maize and may be a foundation for similar studies in other crop species.

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The accessible promoter–mediated supplementary effect of ACE2 provides new insight into the tissue tropism of SARS-CoV-2

10.21203/rs.3.rs-117172/v1 ◽

2020 ◽

Author(s):

Qi Jiang ◽

Guifang Du ◽

Junting Wang ◽

XiaoHan Tang ◽

Xuejun wang ◽

...

Keyword(s):

Cell Lines ◽

High Throughput Sequencing ◽

Chromatin Accessibility ◽

Tissue Tropism ◽

Open Chromatin ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Novel Coronavirus ◽

Accessible Chromatin ◽

Insight Into

Abstract Background：Angiotensin-converting enzyme 2 (ACE2) has been confirmed to be a receptor for the newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, cell surface ACE2 expression is reported to be inconsistent with clinical tissue tropism of SARS-CoV-2, which complicates understanding of the pathogenesis of 2019 novel coronavirus disease (COVID-19). The consumption of ACE2 by internalization and shedding processes may explain this discordance. Results：To understand the discordance between ACE2 expression and the tissue tropism of SARS-CoV-2, we examined the chromatin accessibility of ACE2 promoter in hundreds of tissues and cell lines using public DNase-seq and assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. We find that ACE2 promoter is only accessible in three tissues including lung, large intestine and placenta. Also, we examined tumors tissues and ACE2 promoter is observed accessible in five tumors with reported SARS-CoV-2 susceptibility. We confirmed the susceptibility by performing SARS-CoV-2 pseudovirus infection in several cell lines. Conclusions：We propose that open chromatin at the promoter mediates the ACE2 supplementary effect and ensures the entry of SARS-CoV-2. This hypothesis provides a new view and potential clues for further investigation of COVID-19 pathogenesis.

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ATAC-seq with unique molecular identifiers improves quantification and footprinting

10.1101/2020.10.22.351478 ◽

2020 ◽

Author(s):

Tao Zhu ◽

Keyan Liao ◽

Rongfang Zhou ◽

Chunjiao Xia ◽

Weibo Xie

Keyword(s):

Transcription Factor ◽

High Throughput ◽

High Throughput Sequencing ◽

Pcr Amplification ◽

Chromatin Accessibility ◽

Link Type ◽

Pcr Duplicates ◽

Identify Transcription Factor ◽

Accessible Chromatin ◽

Accurate Quantification

AbstractATAC-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 an 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. In our study, UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq, which helps identify an additional 50% or more of footprints. 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.

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Dynamic chromatin accessibility landscape changes following interleukin-1 stimulation

10.1101/2020.01.29.924878 ◽

2020 ◽

Author(s):

Matt J. Barter ◽

Kathleen Cheung ◽

Julia Falk ◽

Andreas C. Panagiotopoulos ◽

Caitlin Cosimini ◽

...

Keyword(s):

Gene Expression ◽

High Throughput Sequencing ◽

Regulatory Element ◽

Interleukin 1 ◽

Chromatin Modification ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Enhancer Activity ◽

Cytokine Stimulation ◽

Accessible Chromatin

AbstractGenome-wide methods for examining chromatin modification provide detailed information on regulatory regions of the genome. Dynamic modifications of chromatin allow rapid access of the gene regulatory machinery to condensed genomic regions facilitating subsequent gene expression. Inflammatory cytokine stimulation of cells can cause rapid gene expression changes through direct signalling pathway-mediated transcription factor activation and regulatory element binding.Here we used the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess regions of the genome that are differentially accessible following treatment of cells with interleukin-1 (IL-1). We identified 126,483 open chromatin regions, with 241 regions significantly differentially accessible following stimulation, with 64 and 177 more or less accessible, respectively. These differentially accessible regions predominantly correspond to regions of the genome marked as enhancers. Motif searching identified an overrepresentation of a number of transcription factors, most notably RelA in the regions becoming more accessible, with analysis of ChIP-seq data confirmed RelA binding to these regions. A significant correlation in differential chromatin accessibility and gene expression was also observed.Functionality in regulating gene expression was confirmed using CRISPR/Cas9 genome-editing to delete regions for that became more accessible following stimulation in the genes MMP13, IKBKE and C1QTNF1. These same regions were also accessible for activation using a dCas9-transcriptional activator and showed enhancer activity in a cellular model.Together, these data describe and functionally validate a number of dynamically accessible chromatin regions involved in inflammatory signalling.

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