Histone ChIP-seq Research Articles

Identifying dysregulated regions in amyotrophic lateral sclerosis through chromatin accessibility outliers

The high heritability of ALS contrasts with its low molecular diagnosis rate post-genetic testing, pointing to potential undiscovered genetic factors. To aid the exploration of these factors, we introduced EpiOut, an algorithm to identify chromatin accessibility outliers that are regions exhibiting divergent accessibility from the population baseline in a single or few samples. Annotation of accessible regions with histone ChIP-seq and Hi-C indicates that outliers are concentrated in functional loci, especially among promoters interacting with active enhancers. Across different omics levels, outliers are robustly replicated, and chromatin accessibility outliers are reliable predictors of gene expression outliers and aberrant protein levels. When promoter accessibility does not align with gene expression, our results indicate that molecular aberrations are more likely to be linked to post-transcriptional regulation rather than transcriptional regulation. Our findings demonstrate that the outlier detection paradigm can uncover dysregulated regions in rare diseases. EpiOut is available at github.com/uci-cbcl/EpiOut.

Constructing eRNA-mediated gene regulatory networks to explore the genetic basis of muscle and fat-relevant traits in pigs

BackgroundEnhancer RNAs (eRNAs) play a crucial role in transcriptional regulation. While significant progress has been made in understanding epigenetic regulation mediated by eRNAs, research on the construction of eRNA-mediated gene regulatory networks (eGRN) and the identification of critical network components that influence complex traits is lacking.ResultsHere, employing the pig as a model, we conducted a comprehensive study using H3K27ac histone ChIP-seq and RNA-seq data to construct eRNA expression profiles from multiple tissues of two distinct pig breeds, namely Enshi Black (ES) and Duroc. In addition to revealing the regulatory landscape of eRNAs at the tissue level, we developed an innovative network construction and refinement method by integrating RNA-seq, ChIP-seq, genome-wide association study (GWAS) signals and enhancer-modulating effects of single nucleotide polymorphisms (SNPs) measured by self-transcribing active regulatory region sequencing (STARR-seq) experiments. Using this approach, we unraveled eGRN that significantly influence the growth and development of muscle and fat tissues, and identified several novel genes that affect adipocyte differentiation in a cell line model.ConclusionsOur work not only provides novel insights into the genetic basis of economic pig traits, but also offers a generalizable approach to elucidate the eRNA-mediated transcriptional regulation underlying a wide spectrum of complex traits for diverse organisms.

Abstract 3589: Understanding the role of Pax5 in development of taxane-resistant NE-like prostate cancer

Abstract Background: Resistance to the existing Androgen Receptor Signaling Inhibitor (ARSI) therapies led to higher incidences of aggressive therapy-induced neuroendocrine-like prostate cancer (t-NEPC) with predominant small cell-like characteristics, dismal overall survival, and an ultimate resistance to taxane chemotherapies. t-NEPCs are mostly treated with platinum-based drugs with etoposide and/or taxane but have less selectivity and high systemic toxicity, which often limits their clinical and therapeutic potential. During t-NEPC transformation, upon chromatin reprogramming associated with epigenetic alterations and altered transcriptional activity, adenocarcinoma loses its luminal features to adopt neurobasal characteristics. The following study aims to unravel the mechanism of developing t-NEPC taxane resistance upon acquiring such adaptive neuronal characteristics. Objective: The primary objective is to delineate how chromatin modifications along with epigenetic regulation facilitates the adaptive neuronal characteristics and dictates the development of t-NEPC taxane resistance. Methods: We generated various t-NEPC cell lines either by genetic modification (DKD) or therapeutic pressure (C4-2BER). We performed RNA-Seq, ATAC-Seq and acetylated Histone (H3K18 and H3K27) ChIP-Seq in our developed t-NEPC models and compared with adenocarcinoma. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER and performed functional assays. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during t-NEPC transformation. Overlapping RNA seq suggested newly transcribed neuronal genes with higher extent of promoter accessibility. Transcription Factor mapping among newly accessible gene promoters revealed preferential Pax5 binding, whose expression occurs selectively in t-NEPC unlike adenocarcinoma. Pathway analysis of differentially expressed neuronal genes in t-NEPC suggested an involvement of Pax5 in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are essential for strong cellular communication. Furthermore, Pax5 depletion disrupts cellular interaction in t-NEPC, thereby, sensitizing them to taxane therapies. Conclusions: Our study concludes that continuous AR ablation with ARSIs, leads to chromatin alterations with epigenetic changes, thereby allowing Pax5 transcriptional activation in t-NEPC. This altered transcriptional activity promotes gene expression necessary to govern taxane-resistant t-NEPC development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Understanding the role of Pax5 in development of taxane-resistant NE-like prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3589.

Abstract C081: The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma

Abstract Deregulation of proteins involved in chromatin regulation is common in pancreatic ductal adenocarcinoma (PDAC), occurring via both genomic and non-genomic mechanisms. Lysine demethylase 4C (KDM4C) is one of the chromatin-modifying proteins frequently overexpressed across multiple solid cancers. We observed upregulation of Kdm4c protein in a panel of human and murine PDAC cell lines, as well as in PDAC patient samples compared to non-neoplastic controls. CRISPR/Cas9-mediated deletion of KDM4C in human and murine PDAC cells resulted in reduced proliferation and clonogenicity, and increased survival of orthotopically implanted murine PDAC allografts. Notably, in multiple KDM4C-null PDAC clones analyzed by reverse phase protein arrays (RPPA), we observed downregulation of activated mitogen-activated protein kinase (MAP kinase) pathway components (including phospho-MEK and phospho-ERK), which are key effector pathways downstream of mutant KRAS. In vitro propagation of KDM4C-null PDAC lines eventually led to adaptation and restitution of MAP kinase signaling via compensatory upregulation of other KDM4 family members (KDM4A and KDM4B). In order to bypass genetic compensation by KDM4 family members, we utilized a preclinical grade pan-KDM4 inhibitor (TACH107, Tachyon Therapeutics) in PDAC cell lines, which confirmed profound reduction in proliferation and colony formation; in vivo xenograft and allograft studies with TACH107 are now ongoing. To characterize signaling pathways perturbed by KDM4C deregulation, we investigated the transcriptional changes in PDAC cells with and without KDM4C deletion by RNA-seq and integrated these with alterations in H3K9me3 and H3K36me3 marks using histone ChIP-Seq. Regulatory network analysis identified ATF4 as one of the top downregulated transcription factor networks, validating a previously published observation linking KDM4C to amino acid biosynthesis and cell proliferation. Gene set enrichment analysis showed downregulation of RAS signatures, consistent with the RPPA data on attenuation of MAP Kinase pathway. DUSP2, a member of the dual specificity protein phosphatase subfamily that inactivates ERK, is transcriptionally upregulated and has increased H3K36me3 marks near the promoter region in KDM4C-depleted cells, nominating it as a candidate of KDM4C-mediated epigenetic regulation. To further delineate the essential binding partners of KDM4C in chromatin regulation, we have created HA-tagged catalytic domain and reader domain deletion constructs that will be used for immunoprecipitation followed by mass spectrometry analysis. Overall, our data suggests that KDM4C - and other KDM4 family members – regulate MAPK signaling in KRAS-mutant cells, at least partially via attenuation of transcripts encoding specific protein phosphatases. To the best of our knowledge, this is the first demonstration linking the requirement of sustained activity of the KDM4 family of lysine demethylases to MAPK signaling in cancer and presents an opportunity to leverage this oncogenic pathway for therapeutic purposes. Citation Format: Menna-t-Allah Shaheen, Laila Barkoudeh, Sarah Dhebat, Kimal I. Rajapakshe, Bidyut Ghosh, Anirban Maitra. The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C081.

Predmoter-cross-species prediction of plant promoter and enhancer regions.

Identifying cis-regulatory elements (CREs) is crucial for analyzing gene regulatory networks. Next generation sequencing methods were developed to identify CREs but represent a considerable expenditure for targeted analysis of few genomic loci. Thus, predicting the outputs of these methods would significantly cut costs and time investment. We present Predmoter, a deep neural network that predicts base-wise Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) and histone Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) read coverage for plant genomes. Predmoter uses only the DNA sequence as input. We trained our final model on 21 species for 13 of which ATAC-seq data and for 17 of which ChIP-seq data was publicly available. We evaluated our models on Arabidopsis thaliana and Oryza sativa. Our best models showed accurate predictions in peak position and pattern for ATAC- and histone ChIP-seq. Annotating putatively accessible chromatin regions provides valuable input for the identification of CREs. In conjunction with other in silico data, this can significantly reduce the search space for experimentally verifiable DNA-protein interaction pairs. The source code for Predmoter is available at: https://github.com/weberlab-hhu/Predmoter. Predmoter takes a fasta file as input and outputs h5, and optionally bigWig and bedGraph files.

LSD1 Regulates Neurogenesis in Human Neural Stem Cells Through the Repression of Human-Enriched Extracellular Matrix and Cell Adhesion Genes.

Neurogenesis begins with neural stem cells undergoing symmetric proliferative divisions to expand and then switching to asymmetric differentiative divisions to generate neurons in the developing brain. Chromatin regulation plays a critical role in this switch. Histone lysine-specific demethylase LSD1 demethylates H3K4me1/2 and H3K9me1/2 but the mechanisms of its global regulatory functions in human neuronal development remain unclear. We performed genome-wide ChIP-seq of LSD1 occupancy, RNA-seq, and Histone ChIP-seq upon LSD1 inhibition to identify its repressive role in human neural stem cells. Novel downstream effectors of LSD1 were identified, including the Notch signaling pathway genes and human-neural progenitor-enriched extracellular matrix (ECM) pathway/cell adhesion genes, which were upregulated upon LSD1 inhibition. LSD1 inhibition led to decreased neurogenesis, and overexpression of downstream effectors mimicked this effect. Histone ChIP-seq analysis revealed that active and enhancer markers H3K4me2, H3K4me1, and H3K9me1 were upregulated upon LSD1 inhibition, while the repressive H3K9me2 mark remained mostly unchanged. Our work identifies the human-neural progenitor-enriched ECM pathway/cell adhesion genes and Notch signaling pathway genes as novel downstream effectors of LSD1, regulating neuronal differentiation in human neural stem cells.

Analyzing histone ChIP-seq data with a bin-based probability of being signal.

Histone ChIP-seq is one of the primary methods for charting the cellular epigenomic landscape, the components of which play a critical regulatory role in gene expression. Analyzing the activity of regulatory elements across datasets and cell types can be challenging due to shifting peak positions and normalization artifacts resulting from, for example, differing read depths, ChIP efficiencies, and target sizes. Moreover, broad regions of enrichment seen in repressive histone marks often evade detection by commonly used peak callers. Here, we present a simple and versatile method for identifying enriched regions in ChIP-seq data that relies on estimating a gamma distribution fit to non-overlapping 5kB genomic bins to establish a global background. We use this distribution to assign a probability of being signal (PBS) between zero and one to each 5 kB bin. This approach, while lower in resolution than typical peak-calling methods, provides a straightforward way to identify enriched regions and compare enrichments among multiple datasets, by transforming the data to values that are universally normalized and can be readily visualized and integrated with downstream analysis methods. We demonstrate applications of PBS for both broad and narrow histone marks, and provide several illustrations of biological insights which can be gleaned by integrating PBS scores with downstream data types.

Shigella induces epigenetic reprogramming of zebrafish neutrophils.

Trained immunity is a long-term memory of innate immune cells, generating an improved response upon reinfection. Shigella is an important human pathogen and inflammatory paradigm for which there is no effective vaccine. Using zebrafish larvae, we demonstrate that after Shigella training, neutrophils are more efficient at bacterial clearance. We observe that Shigella-induced protection is nonspecific and has differences with training by BCG and β-glucan. Analysis of histone ChIP-seq on trained neutrophils revealed that Shigella training deposits the active H3K4me3 mark on promoter regions of 1612 genes, dramatically changing the epigenetic landscape of neutrophils toward enhanced microbial recognition and mitochondrial ROS production. Last, we demonstrate that mitochondrial ROS plays a key role in enhanced antimicrobial activity of trained neutrophils. It is envisioned that signals and mechanisms we discover here can be used in other vertebrates, including humans, to suggest new therapeutic strategies involving neutrophils to control bacterial infection.

Cis-Cardio: A comprehensive analysis platform for cardiovascular-relavant cis-regulation in human and mouse

Cis-regulatory elements are important molecular switches in controlling gene expression and are regarded as determinant hubs in the transcriptional regulatory network. Collection and processing of large-scale cis-regulatory data are urgent to decipher the potential mechanisms of cardiovascular diseases from a cis-regulatory element aspect. Here, we developed a novel web server, Cis-Cardio, which aims to document a large number of available cardiovascular-related cis-regulatory data and to provide analysis for unveiling the comprehensive mechanisms at a cis-regulation level. The current version of Cis-Cardio catalogs a total of 45,382,361 genomic regions from 1,013 human and mouse epigenetic datasets, including ATAC-seq, DNase-seq, Histone ChIP-seq, TF/TcoF ChIP-seq, RNA polymerase ChIP-seq, and Cohesin ChIP-seq. Importantly, Cis-Cardio provides six analysis tools, including region overlap analysis,element upstream/downstream analysis, transcription regulator enrichment analysis, variant interpretation, andprotein-protein interaction-based co-regulatory analysis. Additionally, Cis-Cardio provides detailed and abundant (epi-) genetic annotations in cis-regulatory regions, such as super-enhancers, enhancers, transcription factor binding sites (TFBSs), methylation sites, common SNPs, risk SNPs, expression quantitative trait loci (eQTLs), motifs,DNase I hypersensitive sites (DHSs), and 3D chromatin interactions. In summary, Cis-Cardio is a valuable resourcefor elucidating and analyzing regulatory cues of cardiovascular-specific cis-regulatory elements. The platform is freely available at http://www.licpathway.net/Cis-Cardio/index.html.

One-carbon metabolism is required for epigenetic stability in the mouse placenta.

One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrr gt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrr gt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrr gt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrr gt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrr gt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance.

Abstract A081: Epigenetic regulation of SPDEF gene in RCC7/T cells, a line of malignant African-American prostate epithelial cells

Abstract Background: Prostate cancer (PCa) in African American (AA) men has an earlier onset, more aggressiveness with higher metastasis and mortality rate than in Caucasian men. SAM-pointed domain-containing Ets-like factor (SPDEF) has been identified as a possible suppressor of metastasis in castration-resistant prostate cancer (CRPC) in earlier investigations. Ongoing studies in our lab are focused on deciphering the role of SPDEF in PCa progression and metastasis with special emphasis on PCa in AA men. We discovered that SPDEF expression is lost in RCC7/T cells, and re-expression of SPDEF limits metastatic properties of these cells, however, the regulatory mechanisms underlying the loss of SPDEF have not been elucidated. In the current study, we investigated the role of epigenetic modulators (DNA methylation and histone modifications) in driving the loss of SPDEF. Methods: We analyzed publicly available data sets from TCGA (GDC TCGA Prostate Cancer, and TCGA Prostate Cancer (PRAD) for DNA methylation using Xena UCSC genome browser (https://xena.ucsc.edu/). We also analyzed histone ChIP-seq data from PCa cell lines using cistrome project (http://cistrome.org/db/#/). RCC7/T cells were grown in DMEM and maintained at 37°C in a humidified incubator. We used bisulfite sequencing (BSP) to examine DNA methylation in SPDEF gene in SPDEF proficient (LNCaP) and deficient (RCC7/T) cells. We also employed the ChIP-qPCR to reveal the active and repressive key histone marks (H3K4me3, H3K27ac, and H3K27me3) across the SPDEF gene. Results: In the clinical cohorts, SPDEF expression showed an inverse correlation with the degree of DNA methylation and expression of the epigenetic writer enzymes such as DNMT1 and EZH2. Our results revealed that the CpG islands in SPDEF gene are hyper-methylated in RCC7/T cells compared to LNCaP cells. Our analysis of the data from the cistrome project revealed an elevated enhancer repressive mark H3K27me3 (marked by EZH2) and a decreased promoter active mark, H3K4me3, in PC3 cells as compared with less metastatic LNCaP cells. Our results from ChIP-qPCR confirmed these findings. Moreover, we observed the methylation profiles in RCC7/T cells were similar to those of PC3 cells. Additionally, combination treatment with 5-aza-2-deoxycytidine (DNMT inhibitor) and GSK-126 (EZH2 inhibitor) increased the PDEF expressions levels and also restricted colony formation, migration and invasion in RCC7/T cells. Conclusion: Overall, these findings reveal an inverse correlation between expression of epigenetic writers (DNMT1 and EZH2) and SPDEF expression in PCa. Our results also suggest that SPDEF expression in PCa is regulated in part by epigenetic modifications, and that combined inhibition of DNMT1 and EZH2 may offer a therapeutic benefit in subsets of PCa patients including in AA men. Acknowledgements: These studies were funded in part by NIH/NCI-7R01CA242839 (HK) and unrestricted funds from the LSU-LCMC Cancer Center, School of Medicine -LSUHSC, New Orleans (HK). Grateful to LCU-LCMC Genomics core for DNA sequencing Citation Format: Mousa Vatanmakanian, Sweaty Koul, Thangavel Chellappagounder, Hari K. Koul. Epigenetic regulation of SPDEF gene in RCC7/T cells, a line of malignant African-American prostate epithelial cells [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A081.

Analyzing histone ChIP-seq data with a bin-based probability of being signal - datasets

Analyzing histone ChIP-seq data with a bin-based probability of being signal - datasets

Abstract A018: KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping

Abstract Triple-negative breast cancer (TNBC) is a subtype accounting for 20-30% all breast cancer cases and is a highly aggressive disease with inferior prognosis. The acquired resistance is a major obstacle for efficient therapy and discovery of novel clinically-actionable targets has become an urgent need. In this study, we carried out an in-depth functional characterization of histone demethylase KDM4C in TNBC and uncovered a novel mechanism underlying KDM4C-driven tumorigenesis. KDM4C is the second most frequently amplified histone demethylase in TNBC. Genetic knockdown or pharmacological inhibition of KDM4C in two amplified basal breast cancer cell lines, SUM149 and HCC1954, drastically suppressed primary tumor growth in vitro and in vivo. Transcriptomic analysis underlined oxidative phosphorylation impairment as functional consequences of KDM4C blockade. Surprisingly, integrating series of histone ChIP-seq and ATAC-seq showed that KDM4C suppression caused accessible chromatin remodeling without substantial changes of its canonical substrates H3K9me3 and H3K36me3. Rather KDM4C loss caused proteolytic cleavage at histone 3 N-terminus (Ala21 site) identified by histone mass spectrometry. Protease inhibitor array pointed out cathepsin L (CTSL) as the endopeptidase mediating this procedure. KDM4C blockade induced CTSL activation and promoted H3 tail clipping at around 30% CTSL binding sites, which was associated with restricted chromatin accessibility and transcriptomic reprogramming. Proteomic interactome profiling revealed that grainyhead like transcription factor 2 (GRHL2) tightly binds to CTSL. In addition, GRHL2 knockout induced nearly complete loss of CTSL chromatin binding, demonstrating its role as a recruiter of CTSL to the chromatin. KDM4C serves as either a direct or indirect suppressor for CTSL-mediated H3 cleavage. The latter one was largely mediated by redox imbalance. Metabolomic profiling showed that KDM4C inhibition strongly dampened intracellular glutathione (GSH) levels and hence produced more reactive oxygen species (ROS) and alleviated mitochondrial respiration. This procedure was grounded on the mechanism that KDM4C blockade decreased expression of GSH synthesis rate-limiting enzyme glutamate-cysteine ligase catalytic subunit (GCLC) via CTSL-mediated H3 tail clipping. Knockout of CTSL rescued KDM4C blockade-associated metabolic dysfunction, confirming KDM4C links redox homeostasis and chromatin remodeling. Finally, we found KDM4C-CTSL-GSH axis was associated with cisplatin resistance in TNBC patients. Co-targeting KDM4C and GSH production efficiently improved cisplatin response in TNBC cells. Our study provided detailed and unbiased evidence supporting a novel non-canonical role of KDM4C in preventing CTSL-mediated histone clipping and maintaining redox balance, opening up possibilities to improve chemotherapy through targeting this circus. Citation Format: Zheqi Li, Guillermo Peluffo, Laura E. Stevens, Xintao Qiu, Shawn B. Egri, Malvina Papanastasiou, Natalie Kingston, Clive S. D'Santos, Eva Papachristou, Kyle Evans, Ji-Heui Seo, Kendell Clement, Daniel Temko, Muhammad Ekram, Anton Simeonov, Stephen C. Kales, Ganesha Rai, Madhu Lal-Nag, David J. Maloney, Ajit Jadhav, Franziska Michor, Alex Meissner, Jason S. Carroll, Matthew L. Freedman, Henry W. Long, Jacob D. Jaffe, Kornelia Polyak. KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping. [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 A018.

Abstract LB037: Chromatin modifications guide Pax5 dependent gene expression in NE like prostate cancer

Abstract Background: Prolonged ablation of androgen receptor pathway in prostate cancer leads to the emergence of therapy resistant neuroendocrine-like (NE-like) aggressive lineage. This NE development is associated with chromatin reprogramming underlying epigenetic alterations and changes in transcriptional network. Clinically, NE development follows a poorer progression with multifaceted therapeutic challenges. Current lack of effective understanding of this NE trans-differentiation process limits the stratification of therapeutic window with further worsened patient management. The following study aims to identify the sequential molecular players responsible for therapy resistant NE like development. Objective: The primary objective of this study is to understand how chromatin modifications along with epigenetic regulation dictates the establishment of NE trans-differentiation process in Prostate Cancer in response to Androgen Receptor blockade therapy. Methods: We generated various NE-like cell lines either by genetic modification or therapeutic selection. We performed RNA Seq, ATAC Seq and acetylated Histone (H3K18 and H3K27) ChIP Seq in our developed NE and adenocarcinoma cell lines. We next compared ATAC Seq and Acetylated histone ChIP Seq with RNA Seq in C4-2B and C4-2BER. Results: Our ATAC Seq and acetylated histone footprints (Ac H3K18 and Ac H3K27 ChIP Seq) analyses revealed an enhanced chromatin accessibility during adenocarcinoma to NE transformation. Overlapping RNA seq results further suggested newly transcribed neuronal genes with higher degrees of promoter accessibility. Transcription Factor scanning analysis among newly active gene promoters revealed preferential binding of Pax5, whose expression has been validated to be selectively occurring in NE lineage unlike adenocarcinoma. Further analysis of Pax5 promoter site suggested that changes in 5-hydroxymethylation pattern during adenocarcinoma to NE transformation guided the recruitment of PBX1 at Pax5 promoter. Validating NE patient expression in silico supported that PBX1/Pax5 expressions are selective in NE lineage development. Conclusions: Our study concludes that specific chromatin alterations guide the recruitment of PBX1 to upregulate Pax5 to maintain a therapy resistant neuronal surrounding in NE-like prostate cancer development. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Juhi Mishra, Dipanwita Das, Kaustubh Datta, Samikshan Dutta. Chromatin modifications guide Pax5 dependent gene expression in NE like prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB037.

Changes Within H3K4me3-Marked Histone Reveal Molecular Background of Neutrophil Functional Plasticity.

Neutrophils are a heterogenous population capable of both antimicrobial functions and suppressor ones, however, no specific pattern of transcription factors controlling this plasticity has been identified. We observed rapid changes in the neutrophil status after stimulation with LPS, pre-activating concentration of TNF-α, or IL-10. Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3 allowed us to identify various transcriptional start sites (TSSs) associated with plasticity and heterogeneity of human neutrophils. Gene Ontology analysis demonstrated great variation within target genes responsible for neutrophil activation, cytokine production, apoptosis, histone remodelling as well as NF-κB transcription factor pathways. These data allowed us to assign specific target genes positioned by H3K4me3-marked histone with a different pattern of gene expression related to NF-κB pathways, apoptosis, and a specific profile of cytokines/chemokines/growth factors realised by neutrophils stimulated by LPS, IL-10, or TNF-α. We discovered IL-10-induced apoptotic neutrophils being transcriptionally active cells capable of switching the profile of cytokines/chemokines/growth factors desired in resolving inflammation via non-canonical NF-κB pathway with simultaneous inhibition of canonical NF-κB pathway. As apoptotic/suppressive neutrophils induced by IL-10 via positioning genes within H3K4me3-marked histone were transcriptionally active, newly described DNA binding sites can be considered as potential targets for immunotherapy.Graphical H3K4me3 histone ChIP-Seq analysis reveals molecular drivers critical for switching neutrophils from their pro- to anti-inflammatory properties.

ERNAs Identify Immune Microenvironment Patterns and Provide a Novel Prognostic Tool in Acute Myeloid Leukemia.

Background: Enhancer RNAs (eRNAs) play an essential role in tumorigenesis as non-coding RNAs transcribed from enhancer regions. However, the landscape of eRNAs in acute myeloid leukemia (AML) and the potential roles of eRNAs in the tumor microenvironment (TME) remain unclear. Method: Gene expression data collected from The Cancer Genome Atlas (TCGA) project were combined with Histone ChIP-seq so as to reveal the comprehensive landscape of eRNAs. Single-sample gene set enrichment analysis algorithm (ssGSEA) and ESTIMATE were employed to enumerate immune cell infiltration and tumor purity. Results: Most prognostic eRNAs were enriched in immune-related pathways. Two distinct immune microenvironment patterns, the immune-active subtype and the immune-resistant subtype, were identified in AML. We further developed an eRNA-derived score (E-score) that could quantify immune microenvironment patterns and predict the response to immune checkpoint inhibitor (ICI) treatment. Finally, we established a prognostic nomogram combining E-score and other clinical features, which showed great discriminative power in both the training set [Harrell’s concordance index (C index): 0.714 (0.651–0.777), p < 0.0001] and validation set [C index: 0.684 (0.614–0.755), p < 0.0001]. Calibration of the nomogram was also validated independently. Conclusion: In this study, we systematically understood the roles of eRNAs in regulating TME diversity and complexity. Moreover, our E-score model provided the first predictive model for ICI treatment in AML.

Decreased expression of transcription factor Homeobox A11 and its potential target genes in bladder cancer

Bladder cancer (BC) ranks as the ninth most commonly diagnosed cancer worldwide. The presence of a transcription factor (TF) has been uncovered as a significant contributor to the pathophysiological changes of cancers. In present study, we elucidated the expression and clinical significance of Homeobox A11 (HOXA11) in BC for the first time, and originally investigated HOXA11 as a TF. Employing in-house immunohistochemistry (IHC), we incorporated 137 BC and 34 non-BC cases to detect the expression of HOXA11 protein in BC tissues. HOXA11-related RNA-sequencing (RNA-seq) expression and RNA microarrays were collected from public databases, the “sva” and “limma” R packages were implemented to integrate and normalize the RNA-seq data and microarrays separately. Integration expression was carried out to further evaluate the HOXA11 expression by utilizing the standard mean difference (SMD). The expression level of HOXA11 in various BC cell lines was also evaluated. We further systematically analyzed the downstream target genes of HOXA11 in BC by utilizing Chromatin Immunoprecipitation Sequencing (ChIP-seq) profiles, differentially expressed genes (DEGs), and HOXA11-related genes. Modification of histone marks on the promoter region of target genes were also discovered by histone ChIP-seq data. Results of the IHC and RNA-seq revealed the protein and mRNA expression of HOXA11 was significantly decreased in BC tissues compared to non-BC tissues (2.98 ± 1.48 vs. 8.23 ± 2.64; 6.87 ± 1.54 vs. 8.38 ± 1.42). Five platforms significantly revealed the down-regulation of HOXA11 expression in BC (GPL96, GPL570, GPL6102, GPL6884, and GPL13497). A similar decreased trend was discovered in BC tissues in expression integration with the incorporated SMD reaching −0.843 (−1.362 ~ −0.325, p = 0.001) and −1.051 (−1.674 ~ −0.428, p = 0.001). Expression of HOXA11 was down-regulated in most of the BC cell lines. COL1A1 was considered as a final HOXA11 target gene and positively related to HOXA11 with the correlation coefficient as 0.584 (95% CI: 0.371–0.739, p < 0.001). HOXA11 regulates COL1A1 expression in BC via H3K27ac modification. The expression of COL1A1 was down-regulated with the SMD reached −0.312 (p < 0.001). In conclusion, HOXA11 expression is markedly decreased and might promote the transcription of COL1A1 to inhibit BC.

Integrative Analysis of Epigenome and Transcriptome Data Reveals Aberrantly Methylated Promoters and Enhancers in Hepatocellular Carcinoma.

DNA methylation is a key transcription regulator, whose aberration was ubiquitous and important in most cancers including hepatocellular carcinoma (HCC). Whole-genome bisulfite sequencing (WGBS) was conducted for comparison of DNA methylation in tumor and adjacent tissues from 33 HCC patients, accompanying RNA-seq to determine differentially methylated region-associated, differentially expressed genes (DMR-DEGs), which were independently replicated in the TCGA-LIHC cohort and experimentally validated via 5-aza-2-deoxycytidine (5-azadC) demethylation. A total of 9,867,700 CpG sites showed significantly differential methylation in HCC. Integrations of mRNA-seq, histone ChIP-seq, and WGBS data identified 611 high-confidence DMR-DEGs. Enrichment analysis demonstrated activation of multiple molecular pathways related to cell cycle and DNA repair, accompanying repression of several critical metabolism pathways such as tyrosine and monocarboxylic acid metabolism. In TCGA-LIHC, we replicated about 53% of identified DMR-DEGs and highlighted the prognostic significance of combinations of methylation and expression of nine DMR-DEGs, which were more efficient prognostic biomarkers than considering either type of data alone. Finally, we validated 22/23 (95.7%) DMR-DEGs in 5-azadC-treated LO2 and/or HepG2 cells. In conclusion, integration of epigenome and transcriptome data depicted activation of multiple pivotal cell cycle-related pathways and repression of several metabolic pathways triggered by aberrant DNA methylation of promoters and enhancers in HCC.

Aiolos regulates eosinophil migration into tissues.

Expression of Ikaros family transcription factor IKZF3 (Aiolos) increases during murine eosinophil lineage commitment and maturation. Herein, we investigated Aiolos expression and function in mature human and murine eosinophils. Murine eosinophils deficient in Aiolos demonstrated gene expression changes in pathways associated with granulocyte-mediated immunity, chemotaxis, degranulation, ERK/MAPK signaling and extracellular matrix organization; these genes had ATAC peaks within 1 kB of the TSS that were enriched for Aiolos binding motifs. Global Aiolos deficiency reduced eosinophil frequency within peripheral tissues during homeostasis; a chimeric mouse model demonstrated dependence on intrinsic Aiolos expression by eosinophils. Aiolos deficiency reduced eosinophil CCR3 surface expression, intracellular ERK1/2 signaling and CCL11-induced actin polymerization, emphasizing an impaired functional response. Aiolos-deficient eosinophils had reduced tissue accumulation in chemokine-, antigen-, and IL-13–driven inflammatory experimental models, all of which at least partially depend on CCR3 signaling. Human Aiolos expression was associated with active chromatin marks enriched for IKZF3, PU.1 and GATA-1 binding motifs within eosinophil-specific histone ChIP-seq peaks. Furthermore, treating the EOL-1 human eosinophilic cell line with lenalidomide yielded a dose-dependent decrease in Aiolos. These collective data indicate that eosinophil homing during homeostatic and inflammatory allergic states is Aiolos dependent, identifying Aiolos as a potential therapeutic target for eosinophilic disease.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.