Chromatin Accessibility Assays Research Articles

A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Benchmarking computational methods for single-cell chromatin data analysis

BackgroundSingle-cell chromatin accessibility assays, such as scATAC-seq, are increasingly employed in individual and joint multi-omic profiling of single cells. As the accumulation of scATAC-seq and multi-omics datasets continue, challenges in analyzing such sparse, noisy, and high-dimensional data become pressing. Specifically, one challenge relates to optimizing the processing of chromatin-level measurements and efficiently extracting information to discern cellular heterogeneity. This is of critical importance, since the identification of cell types is a fundamental step in current single-cell data analysis practices.ResultsWe benchmark 8 feature engineering pipelines derived from 5 recent methods to assess their ability to discover and discriminate cell types. By using 10 metrics calculated at the cell embedding, shared nearest neighbor graph, or partition levels, we evaluate the performance of each method at different data processing stages. This comprehensive approach allows us to thoroughly understand the strengths and weaknesses of each method and the influence of parameter selection.ConclusionsOur analysis provides guidelines for choosing analysis methods for different datasets. Overall, feature aggregation, SnapATAC, and SnapATAC2 outperform latent semantic indexing-based methods. For datasets with complex cell-type structures, SnapATAC and SnapATAC2 are preferred. With large datasets, SnapATAC2 and ArchR are most scalable.

Spatial Multi-omics Reveals the Role of the Wnt Modulator, Dkk2, in Palatogenesis'.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which is the most common of the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study used the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. Although prior research has identified the upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. Moreover, the loss of Pax9 leads to a disruption in the normal osteodifferentiaion of palatal osteogenic mesenchymal cells. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

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

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

Single-Nucleus ATAC-seq for Mapping Chromatin Accessibility in Individual Cells of Murine Hearts.

During the last decade a wide range of single-cell and single-nucleus next-generation sequencing techniques have been developed, which revolutionized detection of rare cell populations, enabling creation of comprehensive cell atlases of complex organs and tissues. State-of-the-art methods do not only allow classical transcriptomics of individual cells but also comprise a number of epigenetic approaches, including assessment of chromatin accessibility by single-nucleus Assay for Transposase Accessible Chromatin ATAC-seq (snATAC-seq). The snATAC-seq assay detects "open chromatin," a term for low nucleosome occupancy of genomic regions, which is a prerequisite for effective transcription factor binding. Information about open chromatin at the single-nucleus level helps to recognize epigenetic changes, sometimes before transcription of respective genes occurs. snATAC-seq detects cellular heterogeneity in otherwise still transcriptionally and/or morphologically homogeneous cell populations. Chromatin accessibility assays may be used to detect epigenetic changes in cardiac lineages during heart development, chromatin landscape changes during aging, and epigenetic alterations in heart diseases. Here, we provide an optimized protocol for snATAC-seq of murine hearts. We describe isolation of single nuclei from snap-frozen hearts, provide hints for preparation of libraries suitable for snATAC-seq next-generation sequencing (NGS) using the Chromium 10× platform, and give general recommendations for downstream analysis using conventional bioinformatic pipelines and packages. The protocol should serve as a beginner's guide to generate high-quality snATAC-seq datasets and to perform chromatin accessibility analysis of individual heart-derived cell nuclei.

Single cell multi‐omics implicates immune and vascular cells in Alzheimer’s etiology

AbstractBackgroundUnderstanding the genetic etiology of Alzheimer’s disease (AD) is critical to inform effective therapies but remains a challenge. Genetic heritability of late‐onset AD is ∼60‐80%, and genome‐wide association studies (GWAS) have uncovered dozens of noncoding single nucleotide polymorphisms (SNPs) that influence AD risk. Two challenges for the functional interpretation of AD genetic risk are (1) determining the cell types in which SNPs operate and (2) identifying the genes they dysregulate to drive AD pathogenesis. Single‐nucleus sequencing studies of human neurons, microglia, and other brain cell types have begun addressing these challenges. Yet, many AD risk SNPs remain unmapped. One possibility is that they are expressed in immunovascular brain cell types largely missed by current methods.MethodHere, we optimized Vessel Isolation and Nuclei Extraction for Sequencing (VINE‐seq) to capture multimodal gene expression and chromatin accessibility assays across all major brain cell types from 30 cognitively normal, MCI, and AD human prefrontal cortex samples.ResultWe discover early gene dysregulation in immune and vascular cell types along AD progression. We highlight the gene regulatory networks mediating this dysregulation and implicate processes associated with AD pathology and cognitive decline. Finally, we map noncoding AD risk variants to diverse immune and vascular cell types, thereby expanding our understanding of AD etiology.ConclusionDysfunction of diverse human brain immune and vascular cell types mediate the genetic risk for Alzheimer’s disease.

Functional genomic studies in diverse populations to characterize risk loci for Alzheimer Disease

AbstractBackgroundAs part of the ADSP Functional Genomics consortium, we aim to unravel ancestry specific effects of genomic regulation of AD loci. African Americans (AA) and Hispanics (HI) are at higher risk for Alzheimer’s disease (AD) compared to non‐Hispanic whites (NHW). Even though AA, HI and other diverse groups make up 40% of the US population, over 90% of participants in large‐scale genomic research studies have been NHW, representing European (EU) ancestry. Recent AD research including diverse groups has shown that genes and variants contributing to disease can have different risk effect dependent on the ancestral background they reside on (i.e. APOE). Together these clearly indicate a need to expand genome‐wide and gene‐specific analyses of genomic organization and regulation for other ancestries contributing to diverse, admixed populations, such as African (AF) or Amerindian (AI) ancestries.MethodWe analyzed interaction of variants in the APOE region with AD risk in WGS of diverse populations to identify modifiers of the APOEɛ4 risk effect. Additionally, to assess overall differences in chromatin regulation genome‐wide across ancestries, we performed bulk and single cell chromatin accessibility (ATAC‐seq) and bulk chromatin interaction (Hi‐C) assays in brains from admixed individuals with high EU, AF (>95%) or AI (>75%) ancestry contribution.ResultWe identified both close‐by and distant modifier variants (in the downstream PSG gene family region) inferring protection against the APOEɛ4 risk on the AF ancestry. Single cell ATAC‐seq data in brain showed higher accessibility of the APOE region and large regions of chr19 in astrocytes, but not microglia, of EU versus AF ancestry. Lastly, bulk Hi‐C analyses in brain strongly supported the hypothesis of ancestry dependent regulation by genome‐wide detection of differential chromatin interactions comparing EU to AF ancestry.ConclusionOur analyses using complementary functional genomic assay methods show clear differences of genome regulation between EU and AF ancestry. Overall, these data support genome regulation to be strongly influenced by ancestral origin. The generated data on samples with high single ancestral contributions will help elucidate risk effects seen for AD related genes in different population groups, as we describe for the APOE region.

Improved quality metrics for association and reproducibility in chromatin accessibility data using mutual information

BackgroundCorrelation metrics are widely utilized in genomics analysis and often implemented with little regard to assumptions of normality, homoscedasticity, and independence of values. This is especially true when comparing values between replicated sequencing experiments that probe chromatin accessibility, such as assays for transposase-accessible chromatin via sequencing (ATAC-seq). Such data can possess several regions across the human genome with little to no sequencing depth and are thus non-normal with a large portion of zero values. Despite distributed use in the epigenomics field, few studies have evaluated and benchmarked how correlation and association statistics behave across ATAC-seq experiments with known differences or the effects of removing specific outliers from the data. Here, we developed a computational simulation of ATAC-seq data to elucidate the behavior of correlation statistics and to compare their accuracy under set conditions of reproducibility.ResultsUsing these simulations, we monitored the behavior of several correlation statistics, including the Pearson’s R and Spearman’s rho coefficients as well as Kendall’s tau and Top–Down correlation. We also test the behavior of association measures, including the coefficient of determination R^2, Kendall’s W, and normalized mutual information. Our experiments reveal an insensitivity of most statistics, including Spearman’s rho, Kendall’s tau, and Kendall’s W, to increasing differences between simulated ATAC-seq replicates. The removal of co-zeros (regions lacking mapped sequenced reads) between simulated experiments greatly improves the estimates of correlation and association. After removing co-zeros, the R^2 coefficient and normalized mutual information display the best performance, having a closer one-to-one relationship with the known portion of shared, enhanced loci between simulated replicates. When comparing values between experimental ATAC-seq data using a random forest model, mutual information best predicts ATAC-seq replicate relationships.ConclusionsCollectively, this study demonstrates how measures of correlation and association can behave in epigenomics experiments. We provide improved strategies for quantifying relationships in these increasingly prevalent and important chromatin accessibility assays.

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned+1 and+2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

MAF amplification licenses ERα through epigenetic remodelling to drive breast cancer metastasis

MAF amplification increases the risk of breast cancer (BCa) metastasis through mechanisms that are still poorly understood yet have important clinical implications. Oestrogen-receptor-positive (ER+) BCa requires oestrogen for both growth and metastasis, albeit by ill-known mechanisms. Here we integrate proteomics, transcriptomics, epigenomics, chromatin accessibility and functional assays from human and syngeneic mouse BCa models to show that MAF directly interacts with oestrogen receptor alpha (ERα), thereby promoting a unique chromatin landscape that favours metastatic spread. We identify metastasis-promoting genes that are de novo licensed following oestrogen exposure in a MAF-dependent manner. The histone demethylase KDM1A is key to the epigenomic remodelling that facilitates the expression of the pro-metastatic MAF/oestrogen-driven gene expression program, and loss of KDM1A activity prevents this metastasis. We have thus determined that the molecular basis underlying MAF/oestrogen-mediated metastasis requires genetic, epigenetic and hormone signals from the systemic environment, which influence the ability of BCa cells to metastasize.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

Zfp296 knockout enhances chromatin accessibility and induces a unique state of pluripotency in embryonic stem cells.

The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.

RELA governs a network of islet-specific metabolic genes necessary for beta cell function

Aims/hypothesisNF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation.MethodsWe generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (βp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (βNEMOKO mice), as well as βA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation–sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme.ResultsRela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in βp65KO islets.Conclusions/interpretationThese data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.Graphical

Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.

Abstract 6267: A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma

Abstract Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homology 2 (EZH2) and its homolog EZH1 are catalytic subunits of polycomb repressive complex 2 (PRC2), which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of its target genes. Although methlytransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. EZH1 also directly binds to chromatin and modulates its condensation. Recent studies have suggested that EZH1 as well as EZH2 played a critical role in T-cell lymphoma such as ATL/L and PTCL, which had high innate EZH1 and increased EZH2 expression upon acquisition of their malignancy. Consequently, dual inhibition of EZH1/2 might induce higher expression of downstream tumor suppressor genes than blocking EZH2 alone, expecting greater activity as an anti-cancer therapy. Herein, we presented a novel and potent EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of both EZH1 and EZH2 with 2.1 and 16 nM of IC50, respectively. Surface plasmon resonance (SPR) assay verified that HM97662 had great binding affinity on EZH1-EED-SUZ12 complex as well as EZH2-EED-SUZ12 complex than competitors. HM97662 also showed broad and strong antiproliferative activity against various T-cell lymphoma cell lines. Representatively in HH cells, HM97662 not only suppressed global tri-methylation of H3K27, but also dose-dependently increased protein expression levels that modulate cell cycle or cancer cell apoptosis. HM97662 exhibited increment of CDKN1A (p21) proteins involved in cell cycle arrest and induced protein levels of cleaved caspase-3 as well as PARP. Additionally, resulting induction of apoptosis by HM97662 in HH cells was confirmed by TUNEL staining. Mechanistically, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH cells. Two cell cycle repressor, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were dose-dependently increased by HM97662. We further conducted chromatin accessibility assay and identified that the chromatin structures of them were loosened and highly transcribed after the treatment of HM97662. Based our in vitro pharmacology data, we evaluated an antitumor activity of HM97662 in EZH1/2 co-expressed HuT-102 T-cell lymphoma cell mouse xenograft model, and daily oral dosing of HM97662 showed potent tumor growth inhibition. In conclusion, the present studies demonstrated that HM97662 has promising prospective for the treatment of patients with T-cell lymphoma. Given that the necessity of new treatment options on T-cell lymphoma, it is urgent to assess the effectiveness of HM97662 in further clinical trials. Citation Format: Jooyun Byun, Seung Hyun Jung, Yu-Yon Kim, Miyoung Lee, Gunwoo Lee, Heesun Moon, Eun Young Lee, Junghwa Park, Seon Yeong Han, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6267.

Correction of transposase sequence bias in ATAC-seq data with rule ensemble modeling.

Chromatin accessibility assays have revolutionized the field of transcription regulation by providing single-nucleotide resolution measurements of regulatory features such as promoters and transcription factor binding sites. ATAC-seq directly measures how well the Tn5 transposase accesses chromatinized DNA. Tn5 has a complex sequence bias that is not effectively scaled with traditional bias-correction methods. We model this complex bias using a rule ensemble machine learning approach that integrates information from many input k-mers proximal to the ATAC sequence reads. We effectively characterize and correct single-nucleotide sequence biases and regional sequence biases of the Tn5 enzyme. Correction of enzymatic sequence bias is an important step in interpreting chromatin accessibility assays that aim to infer transcription factor binding and regulatory activity of elements in the genome.

5P Physiologically relevant treatment models to investigate epigenetic mechanisms driving platinum resistance in ovarian high grade serous carcinoma

The prognosis for patients with platinum-resistant ovarian High Grade Serous Carcinoma (HGSC) remains poor. BriTROC-1 data indicate that genomic alterations alone cannot explain acquired platinum resistance. We investigate epigenetic changes that may drive platinum resistance in HGSC. Using in an in vitro two-dimensional model, we recreated multiple cycles of chemotherapy. We utilised both established cell lines and primary cultures derived from HGSC ascites. Following cell characterisation (p53, PAX8 IHC), carboplatin sensitivity was assessed (sulforhodamine B assay). Cells were then pulsed with four cycles of carboplatin (50μM for 6 hours) with a week of recovery between each cycle. Methylation (Illumina 850k array), transcriptomic (RNAseq) and chromatin accessibility (ATACseq) assays were performed. Cells were also imaged using Stochastic Optical Reconstruction Microscopy. Firstly, we focused on RNAseq analysis to evaluate genes (and related biological processes) that are consistently enriched following carboplatin cycles and the unique genes that are conversely enriched at cycle level. We attempted to identify uniformly up-/down-regulated pathways across the different cultures. These data were compared with the same results obtained from the OvCar4, an established HGSC cell line and IVR01, an in-vivo resistant OvCar4 derivative. The ATACseq and methylation analyses are still ongoing, aiming to identify possible concordant changes in the chromatin accessibility and the methylation patterns that will allow us to clarify the changes observed in transcriptomic. The preliminary data obtained from the RNAseq analysis show that the highest number of enriched genes seems to occur after the first Carboplatin cycle, with a lower number of progressively enriched genes across all other ones. No constant biological processes were identified for the moment, across the different primary cell cultures. Further comparisons will be certainly needed. Understanding the epigenetic landscape of HGSC using physiologically relevant models will allow us to identify possible therapeutic targets that could eventually prevent platinum resistance.

Quality assessment and refinement of chromatin accessibility data using a sequence-based predictive model

Chromatin accessibility assays are central to the genome-wide identification of gene regulatory elements associated with transcriptional regulation. However, the data have highly variable quality arising from several biological and technical factors. To surmount this problem, we developed a sequence-based machine learning method to evaluate and refine chromatin accessibility data. Our framework, gapped k-mer SVM quality check (gkmQC), provides the quality metrics for a sample based on the prediction accuracy of the trained models. We tested 886 DNase-seq samples from the ENCODE/Roadmap projects to demonstrate that gkmQC can effectively identify "high-quality" (HQ) samples with low conventional quality scores owing to marginal read depths. Peaks identified in HQ samples are more accurately aligned at functional regulatory elements, show greater enrichment of regulatory elements harboring functional variants, and explain greater heritability of phenotypes from their relevant tissues. Moreover, gkmQC can optimize the peak-calling threshold to identify additional peaks, especially for rare cell types in single-cell chromatin accessibility data.

Abstract B012: Defining the role of the BAF complex in primary liver cancers

Abstract Incidence and mortality from primary liver cancer are increasing and few effective therapies exist for this deadly disease. Mutations in genes important for chromatin mediated gene regulation commonly occur in both hepatocellular carcinoma and cholangiocarcinoma, the two most common primary liver cancers. Chromatin regulators have been shown to be key modulators of both sensitivity and resistance to therapy response, and therapeutic targeting of chromatin regulators is under development for a wide range of cancers. To uncover chromatin genes that modulate therapy response, we performed epigenome-focused CRISPR screening in the presence of multiple kinases inhibitors (sorafenib, donafenib, cabozantinib). We found that disruption of a specific form of the SWI/SNF chromatin remodeling complex, called BAF, led to therapy resistance. The BAF complex is frequently mutated in both hepatocellular carcinoma and cholangiocarcinoma. Recent studies show that loss of ARID1A and ARID1B, members of the BAF complex, gives rise to liver tumors with morphologies of both hepatocellular carcinoma and cholangiocarcinoma. Critically, because multikinase inhibitors are the most common treatments for advanced hepatocellular carcinoma, our findings suggest patients with BAF mutations would not respond well to these drugs. Using gene expression analysis, genome-wide chromatin localization, and chromatin accessibility assays we are uncovering how altered chromatin state underlies multikinase inhibitor response in BAF mutant tumors. Using high-throughput epigenome focused CRISPR screens we will identify novel modulators of therapy response in liver cancer. Citation Format: Margarita M. Dzama, Deena M. Scoville, Emma D. Bucklan, Jesse R. Raab. Defining the role of the BAF complex in primary liver cancers. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B012.

EPCO-04. TRANSCRIPTIONAL AND CHROMATIN-ACCESSIBILITY HETEROGENEITY IN IDH-MUTANT GLIOMAS

Abstract Diffuse gliomas are a common type of adult brain tumour, comprising about 80% of adult malignant brain tumours, and are incurable. IDH-mutant gliomas, oligodendroglioma and astrocytoma, can be treated initially but eventually recur and are generally fatal. In this study, we profiled and analysed 23 frozen samples from primary and recurrent IDH-mutant glioma patients with single nucleus multiome sequencing (RNA- and ATAC-seq). In total we assayed around 70,000 malignant cells as well as 30,000 cells from the tumour microenvironment. Using a matrix factorisation approach, we characterised biological signals in the malignant cells from each sample, thereby identifying known and novel processes which vary within and recur across samples. Trajectory analysis was used to reconstruct the developmental hierarchy in IDH-mutant glioma and to investigate plasticity at different hierarchy levels, highlighting differences and similarities across patients and cancer types. Finally, master regulators, which drive malignant transcriptional programs were identified, using the complimentary and concurrent RNA and chromatin accessibility assays. This study provides a unique opportunity to investigate the transcriptomic and epigenetic heterogeneity present in gliomas, as well as the events leading to the development of more aggressive and treatment-resistant glioma tumours.