Single-nucleus chromatin accessibility reveals intratumoral epigenetic heterogeneity in IDH1 mutant gliomas

AbstractThe presence of genome-wide DNA hypermethylation is a hallmark of lower grade gliomas (LGG) with isocitrate dehydrogenase (IDH) mutations. Further molecular classification of IDH mutant gliomas is defined by the presence (IDHmut-codel) or absence (IDHmut-noncodel) of hemizygous codeletion of chromosome arms 1p and 19q. Despite the DNA hypermethylation seen in bulk tumors, intra-tumoral heterogeneity at the epigenetic level has not been thoroughly analyzed. To address this question, we performed the first epigenetic profiling of single cells in a cohort of 5 gliomas with IDH1 mutation using single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq). Using the Fluidigm HT IFC microfluidics platform, we generated chromatin accessibility maps from 336 individual nuclei, and identified variable promoter accessibility of non-coding RNAs in LGGs. Interestingly, local chromatin structures of several non-coding RNAs are significant factors that contribute to heterogeneity, and show increased promoter accessibility in IDHmut-noncodel samples. As an example for clinical significance of this result, we identify CYTOR as a poor prognosis factor in gliomas with IDH mutation. Open chromatin assay points to differential accessibility of non-coding RNAs as an important source of epigenetic heterogeneity within individual tumors and between molecular subgroups. Rare populations of nuclei that resemble either IDH mutant molecular group co-exist within IDHmut-noncodel and IDHmut-codel groups, and along with non-coding RNAs may be an important issue to consider for future studies, as they may help guide predict treatment response and relapse.A web-based explorer for the data is available at shiny.turcanlab.org.

Download Full-text

Systematic clustering algorithm for chromatin accessibility data and its application to hematopoietic cells

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008422 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1008422

Author(s):

Azusa Tanaka ◽

Yasuhiro Ishitsuka ◽

Hiroki Ohta ◽

Akihiro Fujimoto ◽

Jun-ichirou Yasunaga ◽

...

Keyword(s):

Data Reduction ◽

Clustering Algorithm ◽

High Throughput Sequencing ◽

Hematopoietic Cells ◽

Cell Types ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Genome Wide ◽

Data Reduction Method ◽

Effective Analysis

The huge amount of data acquired by high-throughput sequencing requires data reduction for effective analysis. Here we give a clustering algorithm for genome-wide open chromatin data using a new data reduction method. This method regards the genome as a string of 1s and 0s based on a set of peaks and calculates the Hamming distances between the strings. This algorithm with the systematically optimized set of peaks enables us to quantitatively evaluate differences between samples of hematopoietic cells and classify cell types, potentially leading to a better understanding of leukemia pathogenesis.

Download Full-text

NEAT-seq: Simultaneous profiling of intra-nuclear proteins, chromatin accessibility, and gene expression in single cells

10.1101/2021.07.29.454078 ◽

2021 ◽

Author(s):

Amy F Chen ◽

Benjamin Parks ◽

Arwa Kathiria ◽

Benjamin Ober-Reynolds ◽

Jorg Goronzy ◽

...

Keyword(s):

T Cells ◽

High Throughput Sequencing ◽

Target Genes ◽

Nuclear Protein ◽

Single Cells ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

T Cell Subsets ◽

Protein Abundance ◽

Joint Measurement

Oligonucleotide-conjugated antibodies have allowed for joint measurement of surface protein abundance and the transcriptome in single cells using high-throughput sequencing. Extending these measurements to gene regulatory proteins in the nucleus would provide a powerful means to link changes in abundance of trans-acting TFs to changes in activity of cis-acting elements and expression of target genes. Here, we introduce Nuclear protein Epitope, chromatin Accessibility, and Transcriptome sequencing (NEAT-seq), a technique to simultaneously measure nuclear protein abundance, chromatin accessibility, and the transcriptome in single cells. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive distinct helper T cell subsets and regulatory T cells (Tregs) and identify examples of TFs with regulatory activity gated by three distinct mechanisms: transcription, translation, and regulation of chromatin binding. Furthermore, we identify regulatory elements and target genes associated with each TF, which we use to link a non-coding GWAS SNP within a GATA motif to both strong allele-specific chromatin accessibility in cells expressing high levels of GATA3 protein, and a putative target gene.

Download Full-text

Single nucleus multi-omics links human cortical cell regulatory genome diversity to disease risk variants

10.1101/2019.12.11.873398 ◽

2019 ◽

Cited By ~ 8

Author(s):

Chongyuan Luo ◽

Hanqing Liu ◽

Fangming Xie ◽

Ethan J. Armand ◽

Kimberly Siletti ◽

...

Keyword(s):

Single Cell ◽

Disease Risk ◽

Single Cells ◽

Cortical Cell ◽

Cell Types ◽

Chromatin Accessibility ◽

Integrative Approach ◽

Risk Variants ◽

Human Frontal Cortex ◽

Single Nucleus

ABSTRACTSingle-cell technologies enable measure of unique cellular signatures, but are typically limited to a single modality. Computational approaches allow integration of diverse single-cell datasets, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells in tissues, we devised single-nucleus methylCytosine, Chromatin accessibility and Transcriptome sequencing (snmC2T-seq) and applied it to post-mortem human frontal cortex tissue. We developed a computational framework to validate fine-grained cell types using multi-modal information and assessed the effectiveness of computational integration methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling prediction of cell types with causal roles in disease.

Download Full-text

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Neuro-Oncology ◽

10.1093/neuonc/noab180.200 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii57-ii57

Author(s):

M Dzwigonska ◽

J Mieczkowski ◽

P Pilanc ◽

S Cyranowski ◽

A Kominek ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

Cancer Cells ◽

Single Cells ◽

Therapy Resistance ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Dependent Manner ◽

Protein Markers ◽

Hypoxic Tumor

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

Download Full-text

Kidney cell type specific changes in the chromatin and transcriptome landscapes following epithelial Hdac1and Hdac2 knockdown

Physiological Genomics ◽

10.1152/physiolgenomics.00102.2021 ◽

2021 ◽

Author(s):

Kelly A. Hyndman ◽

David K Crossman

Keyword(s):

Chromatin Structure ◽

Kidney Cell ◽

Histone Deacetylases ◽

Cell Types ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Electrolyte Balance ◽

Transcript Expression ◽

Single Nucleus ◽

Cell Type Specific

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase-Accessible Chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24-30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.

Download Full-text

Linking transcriptome and chromatin accessibility in nanoliter droplets for single-cell sequencing

10.1101/692608 ◽

2019 ◽

Cited By ~ 1

Author(s):

Song Chen ◽

Blue B Lake ◽

Kun Zhang

Keyword(s):

Mrna Expression ◽

Single Cell ◽

Adult Mouse ◽

Single Cells ◽

Cell Types ◽

Chromatin Accessibility ◽

Single Cell Sequencing ◽

Single Nucleus

Linked profiling of transcriptome and chromatin accessibility from single cells can provide unprecedented insights into cellular status. Here we developed a droplet-based Single-Nucleus chromatin Accessibility and mRNA Expression sequencing (SNARE-seq) assay, that we used to profile neonatal and adult mouse cerebral cortices. To demonstrate the strength of single-cell dual-omics profiling, we reconstructed transcriptome and epigenetic landscapes of cell types, uncovered lineage-specific accessible sites, and connected dynamics of promoter accessibility with transcription during neurogenesis.

Download Full-text

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.

Download Full-text

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

10.1101/138685 ◽

2017 ◽

Cited By ~ 5

Author(s):

Stephen J. Clark ◽

Ricard Argelaguet ◽

Chantriolnt-Andreas Kapourani ◽

Thomas M. Stubbs ◽

Heather J. Lee ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Single Cells ◽

Embryonic Stem ◽

Transcriptome Profiling ◽

Chromatin Accessibility ◽

Open Chromatin ◽

Dynamic Coupling ◽

Single Cell Sequencing ◽

Molecular Layers

AbstractParallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

Download Full-text

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.

Download Full-text

Uniaxial Cyclic Stretching Promotes Chromatin Accessibility of Gene Loci Associated With Mesenchymal Stem Cells Morphogenesis and Osteogenesis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.664545 ◽

2021 ◽

Vol 9 ◽

Author(s):

Duo Zhang ◽

Ran Zhang ◽

Xiaoyuan Song ◽

Karen Chang Yan ◽

Haiyi Liang

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

High Throughput Sequencing ◽

Chromatin Accessibility ◽

Cyclic Stretch ◽

Marker Genes ◽

Open Chromatin ◽

Cell Morphogenesis ◽

Cyclic Stretching ◽

Cell Reorientation

It has been previously demonstrated that uniaxial cyclic stretching (UCS) induces differentiation of mesenchymal stem cells (MSCs) into osteoblasts in vitro. It is also known that interactions between cells and external forces occur at various aspects including cell–matrix, cytoskeleton, nucleus membrane, and chromatin. However, changes in chromatin landscape during this process are still not clear. The present study was aimed to determine changes of chromatin accessibility under cyclic stretch. The influence of cyclic stretching on the morphology, proliferation, and differentiation of hMSCs was characterized. Changes of open chromatin sites were determined by assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Our results showed that UCS induced cell reorientation and actin stress fibers realignment, and in turn caused nuclear reorientation and deformation. Compared with unstrained group, the expression of osteogenic and chondrogenic marker genes were the highest in group of 1 Hz + 8% strain; this condition also led to lower cell proliferation rate. Furthermore, there were 2022 gene loci with upregulated chromatin accessibility in 1 Hz + 8% groups based on the analysis of chromatin accessibility. These genes are associated with regulation of cell morphogenesis, cell–substrate adhesion, and ossification. Signaling pathways involved in osteogenic differentiation were found in up-regulated GO biological processes. These findings demonstrated that UCS increased the openness of gene loci associated with regulation of cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, the findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation, and deformation. Our study may provide reference for directed differentiation of stem cells induced by mechanical microenvironments.

Download Full-text