Deciphering cell types by integrating scATAC-seq data with genome sequences.

The single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) technology provides insight into gene regulation and epigenetic heterogeneity at single-cell resolution, but cell annotation from scATAC-seq remains challenging due to high dimensionality and extreme sparsity within the data. Existing cell annotation methods mostly focus on the cell peak matrix without fully utilizing the underlying genomic sequence. Here we propose a method, SANGO, for accurate single-cell annotation by integrating genome sequences around the accessibility peaks within scATAC data. The genome sequences of peaks are encoded into low-dimensional embeddings, and then iteratively used to reconstruct the peak statistics of cells through a fully connected network. The learned weights are considered as regulatory modes to represent cells, and utilized to align the query cells and the annotated cells in the reference data through a graph transformer network for cell annotations. SANGO was demonstrated to consistently outperform competing methods on 55 paired scATAC-seq datasets across samples, platforms and tissues. SANGO was also shown to be able to detect unknown tumor cells through attention edge weights learned by the graph transformer. Moreover, from the annotated cells, we found cell-type-specific peaks that provide functional insights/biological signals through expression enrichment analysis, cis-regulatory chromatin interaction analysis and motif enrichment analysis.

Profiling the Epigenetic Landscape of the Tumor Microenvironment Using Chromatin Immunoprecipitation Sequencing.

Cancer cells within a tumor exhibit phenotypic plasticity that allows adaptation and survival in hostile tumor microenvironments. Reprogramming of epigenetic landscapes can support tumor progression within a specific microenvironment by influencing chromatin accessibility and modulating cell identity. The profiling of epigenetic landscapes within various tumor cell populations has significantly improved our understanding of tumor progression and plasticity. This protocol describes an integrated approach using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) optimized to profile genome-wide post-translational modifications of histone tails in tumors. Essential tools amenable to ChIP-seq to isolate tumor cell populations of interest from the tumor microenvironment are also presented to provide a comprehensive approach to perform heterogeneous epigenetic landscape profiling of the tumor microenvironment.

The chromatin accessibility landscape in the dental pulp of mouse molars and incisors.

The dental pulp is a promising source of progenitor cells for regenerative medicine. The natural function of dental pulp is to produce odontoblasts to generate reparative dentin. Stem cells within the pulp tissue originate from the migrating neural crest cells and possess mesenchymal stem cell properties with the ability to differentiate into multiple lineages. To elucidate the transcriptional control mechanisms underlying cell fate determination, we compared the transcriptome and chromatin accessibility in primary dental pulp tissue derived from 5-6-day-old mice. Using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we correlated gene expression with chromatin accessibility. We found that the majority of ATAC-seq peaks were concentrated at genes associated with development and cell differentiation. Most of these genes were highly expressed in the mouse dental pulp. Surprisingly, we uncovered a group of genes encoding master transcription factors that were not expressed in the dental pulp but retained open chromatin states. Within this group, we identified key developmental genes important for specification of the neural crest, adipocyte, neural, myoblast, osteoblast and hepatocyte lineages. Collectively, our results uncover a complex relationship between gene expression and the chromatin accessibility landscape in the mouse dental pulp.

The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2.

AIMS: Understanding human neurogenesis is critical toward regenerative medicine for neurodegeneration. However, little is known how neural differentiation is regulated by DEAD box-containing RNA helicases, which comprise a diverse class of RNA remodeling enzymes. MATERIALS AND METHODS: ChIP-seq was utilized to identify binding sites of DDX5 and DDX17 in both human pluripotent stem cell (hPSC) line NTERA2 and their retinoic acid-induced neural derivatives. RNA-seq was used to elucidate genes differentially expressed upon depletion of DDX5 and DDX17. Neurosphere assay, flow cytometry, and immunofluorescence staining were performed to test the effect of depletion of the two RNA helicases in neural differentiation. KEY FINDINGS: We show here that expression of DDX5 and DDX17 is abundant throughout neural differentiation of NTERA2, and is mostly localized within the nucleus. The two RNA helicases occupy chromatin genome-wide at regions associated with neurogenesis-related genes in both hPSCs and their neural derivatives. Further, both DDX5 and DDX17 are mutually required for controlling transcriptional expression of these genes, but are not important for maintenance of stem cell state of hPSCs. In contrast, they facilitate early neural differentiation of hPSCs, generation of neurospheres from the stem cells, and transcriptional expression of key neurogenic transcription factors such as SOX1 and PAX6 during neural differentiation. Importantly, DDX5 and DDX17 are critical for differentiation of hPSCs toward NESTIN- and TUBB3-positive cells, which represent neural progenitors and mature neurons, respectively. SIGNIFICANCE: Collectively, our findings suggest the role of DDX5 and DDX17 in transcriptional regulation of genes involved in neurogenesis, and hence in neural differentiation of hPSCs.

Dependence on Bcl6 and Blimp1 drive distinct differentiation of murine memory and follicular helper CD4+ T cells.

During the immune response, CD4+ T cells differentiate into distinct effector subtypes, including follicular helper T (Tfh) cells that help B cells, and into memory cells. Tfh and memory cells are required for long-term immunity; both depend on the transcription factor Bcl6, raising the question whether they differentiate through similar mechanisms. Here, using single-cell RNA and ATAC sequencing, we show that virus-responding CD4+ T cells lacking both Bcl6 and Blimp1 can differentiate into cells with transcriptomic, chromatin accessibility, and functional attributes of memory cells but not of Tfh cells. Thus, Bcl6 promotes memory cell differentiation primarily through its repression of Blimp1. These findings demonstrate that distinct mechanisms underpin the differentiation of memory and Tfh CD4+ cells and define the Bcl6-Blimp1 axis as a potential target for promoting long-term memory T cell differentiation.

Kidney cell type-specific changes in the chromatin and transcriptome landscapes following epithelial Hdac1 and Hdac2 knockdown.

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 and 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.

Clipper: p-value-free FDR control on high-throughput data from two conditions.

High-throughput biological data analysis commonly involves identifying features such as genes, genomic regions, and proteins, whose values differ between two conditions, from numerous features measured simultaneously. The most widely used criterion to ensure the analysis reliability is the false discovery rate (FDR), which is primarily controlled based on p-values. However, obtaining valid p-values relies on either reasonable assumptions of data distribution or large numbers of replicates under both conditions. Clipper is a general statistical framework for FDR control without relying on p-values or specific data distributions. Clipper outperforms existing methods for a broad range of applications in high-throughput data analysis.

Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia.

Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

p53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.

p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice.

The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(4;11).

Leukemia patients bearing the t(4;11)(q21;q23) translocations can be divided into two subgroups: those expressing both reciprocal fusion genes, and those that have only the MLL-AF4 fusion gene. Moreover, a recent study has demonstrated that patients expressing both fusion genes have a better outcome than patients that are expressing the MLL-AF4 fusion protein alone. All this may point to a clonal process where the reciprocal fusion gene AF4-MLL could be lost during disease progression, as this loss may select for a more aggressive type of leukemia. Therefore, we were interested in unraveling the decisive role of the AF4-MLL fusion protein at an early timepoint of disease development. We designed an experimental model system where the MLL-AF4 fusion protein was constitutively expressed, while an inducible AF4-MLL fusion gene was induced for only 48 h. Subsequently, we investigated genome-wide changes by RNA- and ATAC-Seq experiments at distinct timepoints. These analyses revealed that the expression of AF4-MLL for only 48 h was sufficient to significantly change the genomic landscape (transcription and chromatin) even on a longer time scale. Thus, we have to conclude that the AF4-MLL fusion protein works through a hit-and-run mechanism, probably necessary to set up pre-leukemic conditions, but being dispensable for later disease progression.

Genetic and epigenetic basis of hepatoblastoma diversity.

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq.

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost 'capture' method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.

ChIP-GSM: Inferring active transcription factor modules to predict functional regulatory elements.

Transcription factors (TFs) often function as a module including both master factors and mediators binding at cis-regulatory regions to modulate nearby gene transcription. ChIP-seq profiling of multiple TFs makes it feasible to infer functional TF modules. However, when inferring TF modules based on co-localization of ChIP-seq peaks, often many weak binding events are missed, especially for mediators, resulting in incomplete identification of modules. To address this problem, we develop a ChIP-seq data-driven Gibbs Sampler to infer Modules (ChIP-GSM) using a Bayesian framework that integrates ChIP-seq profiles of multiple TFs. ChIP-GSM samples read counts of module TFs iteratively to estimate the binding potential of a module to each region and, across all regions, estimates the module abundance. Using inferred module-region probabilistic bindings as feature units, ChIP-GSM then employs logistic regression to predict active regulatory elements. Validation of ChIP-GSM predicted regulatory regions on multiple independent datasets sharing the same context confirms the advantage of using TF modules for predicting regulatory activity. In a case study of K562 cells, we demonstrate that the ChIP-GSM inferred modules form as groups, activate gene expression at different time points, and mediate diverse functional cellular processes. Hence, ChIP-GSM infers biologically meaningful TF modules and improves the prediction accuracy of regulatory region activities.

A Panel of rSNPs Demonstrating Allelic Asymmetry in Both ChIP-seq and RNA-seq Data and the Search for Their Phenotypic Outcomes through Analysis of DEGs.

Currently, the detection of the allele asymmetry of gene expression from RNA-seq data or the transcription factor binding from ChIP-seq data is one of the approaches used to identify the functional genetic variants that can affect gene expression (regulatory SNPs or rSNPs). In this study, we searched for rSNPs using the data for human pulmonary arterial endothelial cells (PAECs) available from the Sequence Read Archive (SRA). Allele-asymmetric binding and expression events are analyzed in paired ChIP-seq data for H3K4me3 mark and RNA-seq data obtained for 19 individuals. Two statistical approaches, weighted z-scores and predicted probabilities, were used to improve the efficiency of finding rSNPs. In total, we identified 14,266 rSNPs associated with both allele-specific binding and expression. Among them, 645 rSNPs were associated with GWAS phenotypes; 4746 rSNPs were reported as eQTLs by GTEx, and 11,536 rSNPs were located in 374 candidate transcription factor binding motifs. Additionally, we searched for the rSNPs associated with gene expression using an SRA RNA-seq dataset for 281 clinically annotated human postmortem brain samples and detected eQTLs for 2505 rSNPs. Based on these results, we conducted Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and constructed the protein-protein interaction networks to represent the top-ranked biological processes with a possible contribution to the phenotypic outcome.

Glucocorticoid receptor wields chromatin interactions to tune transcription for cytoskeleton stabilization in podocytes.

Elucidating transcription mediated by the glucocorticoid receptor (GR) is crucial for understanding the role of glucocorticoids (GCs) in the treatment of diseases. Podocyte is a useful model for studying GR regulation because GCs are the primary medication for podocytopathy. In this study, we integrated data from transcriptome, transcription factor binding, histone modification, and genome topology. Our data reveals that the GR binds and activates selective regulatory elements in podocyte. The 3D interactome captured by HiChIP facilitates the identification of remote targets of GR. We found that GR in podocyte is enriched at transcriptional interaction hubs and super-enhancers. We further demonstrate that the target gene of the top GR-associated super-enhancer is indispensable to the effective functioning of GC in podocyte. Our findings provided insights into the mechanisms underlying the protective effect of GCs on podocyte, and demonstrate the importance of considering transcriptional interactions in order to fine-map regulatory networks of GR.

ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress.

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

CRISPRi-mediated functional analysis of NKX2-1-binding sites in the lung.

The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense (SFTPB, LAMP3, SFTPA1, SFTPA2) and lung tumorigenesis (MYBPH, LMO3, CD274/PD-L1). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.

Pancreatic cancer prognosis is predicted by an ATAC-array technology for assessing chromatin accessibility.

Unlike other malignancies, therapeutic options in pancreatic ductal adenocarcinoma (PDAC) are largely limited to cytotoxic chemotherapy without the benefit of molecular markers predicting response. Here we report tumor-cell-intrinsic chromatin accessibility patterns of treatment-naïve surgically resected PDAC tumors that were subsequently treated with (Gem)/Abraxane adjuvant chemotherapy. By ATAC-seq analyses of EpCAM+ PDAC malignant epithelial cells sorted from 54 freshly resected human tumors, we show here the discovery of a signature of 1092 chromatin loci displaying differential accessibility between patients with disease free survival (DFS) < 1 year and patients with DFS > 1 year. Analyzing transcription factor (TF) binding motifs within these loci, we identify two TFs (ZKSCAN1 and HNF1b) displaying differential nuclear localization between patients with short vs. long DFS. We further develop a chromatin accessibility microarray methodology termed "ATAC-array", an easy-to-use platform obviating the time and cost of next generation sequencing. Applying this methodology to the original ATAC-seq libraries as well as independent libraries generated from patient-derived organoids, we validate ATAC-array technology in both the original ATAC-seq cohort as well as in an independent validation cohort. We conclude that PDAC prognosis can be predicted by ATAC-array, which represents a low-cost, clinically feasible technology for assessing chromatin accessibility profiles.

Oocyte-specific linker histone H1foo interacts with Esrrb to induce chromatin decondensation at specific gene loci.

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Although, from various previous studies, evidence related to H1foo function in chromatin structures is being accumulated, the distribution of H1foo at the target gene loci in a genome-wide manner and the molecular mechanism of H1foo-dependent chromatin architecture remain unclear. In this study, we aimed to identify the target loci and the physiological factor bound to H1foo at the loci. Chromatin immunoprecipitation sequencing analysis of H1foo-overexpressing mouse embryonic stem cells showed that H1foo is enriched around the transcriptional start sites of genes such as oocyte-specific genes and that the chromatin structures at these regions were relaxed. We demonstrated that H1foo was physiologically bound to the nuclear receptor estrogen-related receptor beta (Esrrb), and Esrrb was necessary for H1foo activity of chromatin decondensation at the target loci. The specific localization and interaction with Esrrb were validated in endogenous H1foo of oocytes. Overall, H1foo induces chromatin decondensation in a locus-specific manner and this function is achieved by interacting with Esrrb.

A High-Throughput Chromatin Immunoprecipitation Sequencing Approach to Study the Role of MYC on the Epigenetic Landscape.

MYC is a transcription factor playing multiple functions both in physiological and pathological settings. Biochemical characterizations, combined with the analyses of MYC chromatin binding, have shown that its pleiotropic activity depends on the chromatin context and its protein-protein interactions with different cofactors. In order to determine the contribution of MYC in a certain biological condition, it would be relevant to analyze the concomitant binding of MYC and its associated proteins, in relationship to the chromatin environment. To this end, we here provide a simple method to parallel map the genome-wide binding of MYC-associated proteins, together with the chromatin profiling of multiple histone modifications. We detail the procedure to perform high-throughput ChIP-seq (HT-ChIP-seq) with a variety of biological samples. In addition, we describe simple bioinformatic steps to determine the distribution of MYC binding with respect to the chromatin context and the association of its cofactors. The described approach will permit the reproducible characterization of MYC activity in different biological contexts.