Analysis Of Histone Modifications Research Articles

Genome-wide analysis of histone modifications can contribute to the identification of candidate cis-regulatory regions in the threespine stickleback fish

BackgroundCis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq).ResultsUsing the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions.ConclusionsThus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

F.2 Comprehensively mapping transcriptionally relevant histone modifications in aggressive meningioma leads to novel biologic insights and therapeutic vulnerabilities

Background: We recently identified four molecular subgroups of meningioma with distinct biology and outcomes. While two (MG3/MG4) are associated with poor outcome, they display divergent transcriptional profiles (enriched in metabolic and cell cycling pathways, respectively) and therapeutic vulnerabilities (MG3 has no clear treatment target). We sought to understand drivers of these key differences at a chromatin level. Methods: We profiled MG3/MG4 meningiomas for common histone marks H3K27me3, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, and H3K36me3. Multiple computational approaches were used to compare MG3 and MG4 tumours including superenhancer ranking, differential binding analysis, and unsupervised clustering. Results: Our cohort includes 11-20 meningiomas per histone mark. Clustering revealed striking separation of subgroups based on multiple histone marks, particularly H3K36me3. FOXC1, a known driver of the epithelial to mesenchymal transition, was identified as a recurrent superenhancer in both groups, whereas MG3-specific superenhancers mapped to immune regulatory networks. Integrated differential binding analysis confirmed an immune-rich microenvironment in MG3 tumours driven by multiple histone marks, suggesting a role for targeting novel immune checkpoint genes CD84 and CD48. Conclusions: This study is the first to apply integrated analysis of multiple histone modifications to aggressive meningioma. We further characterize MG3 tumours by identifying an epigenetically-driven immune phenotype and propose novel treatment targets.

Abstract PS06-07: Liquid biopsy determination of HER2 status in breast cancer: results from a novel epigenomic platform

Abstract Background: Tumor HER2 status remains a critical predictive biomarker for patients with breast cancer given the availability of highly effective anti-HER2 therapies. However, determination of HER2 status relies on a complex, tissue-dependent algorithm optimized for extremes of the phenotype – HER2+ vs HER2- status. Given the challenges in reliably obtaining informative tissue biopsies as disease progresses, the new therapeutic relevance of HER2-low breast cancer (IHC 1+ and FISH-negative IHC 2+), and the undependability of biomarkers in this range, better approaches are needed. Epigenomic signatures detectable in cell-free tumor DNA (cfDNA) may provide a minimally invasive, reliable determination of HER2 status. Here, we present a multimodal epigenomic liquid biopsy platform that could offer a minimally invasive alternative to tissue-based determination of transcriptionally regulated targets such as HER2. Methods: We identified patients with metastatic breast cancer (n=20) that were HER2-positive with IHC 3+ (n=6) or HER2-negative with IHC 0 (n=14) based on ASCO/CAP guidelines who underwent research biopsies at the Dana-Farber Cancer Institute from 2012-2023. Study participants had undergone concurrent blood sampling collected in Streck Tubes, processed as plasma, and stored at -80ºC. We applied a novel, multimodal epigenomic assay to profile tumor-derived gene regulatory programs from 1mL plasma. This genome-wide approach analyzed histone modifications associated with active gene promoters and enhancers, and DNA methylation. Classifier feature selection was derived from epigenomically profiled breast cancer cell lines. Final classifier performance was cross validated using a standard Leave one Out (LoO) scheme. Results: Our cross-validated classifier yielded an area under the ROC curve (AUC) of 0.91 in the 20 samples tested. When including only samples with detectable ctDNA as assessed by low pass whole genome sequencing (ichorCNA algorithm) (N=13), our classifier had an AUC of 0.98. Using regularized regression approach, we identified 33 loci with the highest predictive value; 8 loci were within the ERBB2 amplicon on chromosome 17, while the remaining 25 loci were in other genomic regions. We will present results from this and an expanded cohort, including participants with HER2 IHC 1+ and 2+ disease. Conclusions: We present a novel, minimally invasive HER2 classifier leveraging epigenomic information from cell-free tumor DNA from 1 mL plasma. If this approach remains robust in accurately classifying additional HER2 states, it could address the limitations of current tissue-based testing. Integration of this epigenomic platform in prospective interventional studies could support the development of novel and improved predictive classifiers for response to HER2-directed therapy. Citation Format: Heather Parsons, Sylvan Baca, Anthony D'Ippolito, Hyun-Hwan Jeong, Kalie Smith, Paolo Tarantino, Melissa Hughes, Kerry Sendrick, Corrie Painter, Ji-Heui Seo, Elizabeth Grant, Jamey Guess, Matthew Davidsohn, Gitanjali Lakshminarayanan, Sarah Strauss, Hunter Savignano, John Canniff, Brad Fortunato, Matthew Eaton, Toni Choueiri, Sara Tolaney, Matthew Freedman, Nancy Lin. Liquid biopsy determination of HER2 status in breast cancer: results from a novel epigenomic platform [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS06-07.

Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencingof gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPRinterference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

Abstract 7023: Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids

Abstract Introduction/Background: Epigenetic alterations, including histone modifications and DNA methylation, drive treatment resistance and lethal prostate cancer. However, epigenetic assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. While identifying these alterations is possible in cell lines, these analytes are more difficult to follow in clinical settings. Patient-derived organoids have emerged as viable intermediaries to investigate the changing chromatin landscape during the progression toward treatment-emergent prostate cancer. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input prostate cancer spheroids. Methods: The Oil Immersed Lossless Total Analysis System (OIL-TAS) is an exclusive liquid repellency (ELR) platform that passes magnetic bead-bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of LNCaP prostate cancer cell lines harvested from either 2D cell culture or 3D spheroids was performed to evaluate the sensitivity and specificity of this integrated assay. Results: The macroscale CUT&Tag assay has been performed with inputs of 25,000 nuclei from LNCaP spheroids with greater yields than similar inputs from 2D LNCaP cell culture. Yield increases have also been seen when performing the assay with inputs <10,000 nuclei in microscale on the OIL-TAS relative to the macroscale. Utilizing the OIL-TAS platform with spheroid inputs will allow for more sensitive investigation of histone modifications. Conclusions: Integration of 3D, humanized models of tumor growth into the CUT&Tag assay will enable discovery of differential epigenetic regulation within patient-derived organoids or complex co-culture studies. Establishing baseline epigenetic landscapes among these intermediary models will enable a new platform for drug testing. Utilization of the OIL-TAS platform will allow for miniaturization of the assay down to 1,000 cell inputs and enable multiparametric RNA-seq, EM-seq, and CUT&Tag-seq of the same patient samples for longitudinal study. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Erika Heninger, Xavier Hazelberg, Cole Gilsdorf, Shuang G. Zhao, Duane S. Juang, David J. Beebe, Joshua M. Lang. Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids [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 7023.

Genome-Wide Identification of Osmanthus fragrans Histone Modification Genes and Analysis of Their Expression during the Flowering Process and under Azacytidine and Ethylene Treatments

Histone acetylation and methylation, governed by various histone modification (HM) gene families, are vital for plant biological processes. However, there are limited studies that have explored HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans). We performed genome-wide search and identified 208 OfHMs, encompassing 81 histone methyltransferases (OfHMTs), 51 histone demethylases (OfHDMs), 49 histone acetyltransferases (OfHATs) and 27 histone deacetylases (HDACs). Our comprehensive analysis covered chromosome locations, gene structures, conserved domains, cis-acting elements, phylogenetic comparisons, protein interaction networks and functional enrichment pathways for these gene families. Additionally, tandem and fragment replications were unveiled as contributors to the expansion of OfHMs, with some genes exhibiting positive selection. Furthermore, we examined OfHM expression profiles across various tissues and flowering stages, and under 5′-azacytidine (Aza) and ethylene treatments. Most OfHMs displayed heightened expression in leaves, and were downregulated during the flower opening and senescence stages, including OfPRMTs, OfHDTs, OfHDAs, OfSRTs, OfJMJs and OfHAGs; 75.86% and 80.77% of the differentially expressed OfHMs were upregulated after Aza and ethylene treatments, including OfHAGs, OfHDAs and OfSDGs. This study offers a comprehensive analysis of the OfHM gene family, which indicated their potential involvement in ethylene and Aza responses, and in the flowering process. These findings provide valuable insights into the role of OfHMs in flowering and stress responses.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Evaluating histone modification analysis of individual preimplantation embryos

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT).ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing.ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.

In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.

Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described. This technique allows investigators to examine histone and co-factor interactions with unintegrated viral DNAs as well as to analyze histone modifications in general or in a kinetic fashion at various time points during viral infection.

Genomic and epigenomic insights into the mechanism of cold response in upland cotton (Gossypium hirsutum)

Functional genome research, including gene transcriptional and posttranslational modifications of histones, can benefit greatly from a high-quality genome assembly. Histone modification plays a significant role in modulating the responses to abiotic stress in plants. However, there are limited reports on the involvement of dynamic changes in histone modification in cold stress response in upland cotton. In this study, the genome of an elite accession, YM11, with considerable cold stress tolerance was de novo assembled, which yielded a genome of 2343.06 Mb with a contig N50 of 88.96 Mb, and a total of 73,821 protein-coding gene models were annotated. Comparisons among YM11 and five Gossypium allopolyploid cotton assemblies highlighted a large amount of structural variations and presence/absence variations. We analyzed transcriptome and metabolome changes in YM11 seedlings subjected to cold stress. Using the CUT&Tag method, genome-wide H3K4me3 and H3K9ac modification patterns and effect of histone changes on gene expression were profiled during cold stress. Significant and consistently changing histone modifications and the gene expressions were screened, of which transcription factors (TFs) were highlighted. Our results suggest a positive correlation between the changes in H3K4me3, H3K9ac modifications and cold stress-responsive gene activation. This genome assembly and comprehensive analysis of genome-wide histone modifications and gene expression provide insights into the genomic variation and epigenetic responses to cold stress in upland cotton.

Single-cell joint analysis of gene expression and histone modifications in droplets.

Single-cell joint analysis of gene expression and histone modifications in droplets.

Histone proteomics implicates H3K36me2 and its regulators in mouse embryonic stem cell pluripotency exit and lineage choice

Abstract Objectives Gene expression changes during embryonic stem cell (ESC) differentiation is regulated by epigenetic mechanisms. Understanding these can help uncover how cell fate decisions are made during early embryonic development. Comparison of chromatin of ESCs with lineage-committed cells can implicate chromatin factors functional in exit from pluripotency and the choice of proper lineages. Therefore, we quantitatively analyzed histone modifications in mouse ESC differentiation towards neuroectoderm and endoderm. Methods We cultured mouse ESCs (mESCs) and differentiated them towards neuroectoderm or endoderm lineages and performed mass spectrometry on total histones. Subsequent Western blots verified significantly altered H3K36me2. RT-qPCR analyses showed changes in H3K36-specific methyltransferases, demethylases and readers at mESC stage or during neuroectoderm/endoderm commitment. Results We presented quantitative histone modification levels in mESCs and lineage-committed cells. H3K36me2 increased specifically in neuroectoderm compared to mESCs or endoderm-committed cells. Regulation of H3K36 methylation might have a role in pluripotency exit and/or differentiation. Nsd2, Dnmt3b and Zmynd11 increased during differentiation regardless of lineage. Conversely, mESCs had higher Kdm4c and Msh6 expression than differentiated cells. Comparing neuroectoderm and endoderm-committed cells, we revealed Nsd1, Setd5 and Dnmt3a had lineage specific expression pattern. Conclusions Our results show quantitative changes in histone modifications during mESC lineage commitment and implicate H3K36me2 regulation for not only pluripotency exit but also lineage choice. Its regulatory proteins show stage (mESC vs. committed) or lineage (neuroectoderm vs. endoderm) dependent expression changes. Further work will be needed to discover their possible involvement in cell fate decisions and target genes.

A Rapid and Efficient Method for the Extraction of Histone Proteins.

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.

Histone modification analysis reveals common regulators of gene expression in liver and blood stage merozoites of Plasmodium parasites

Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages.

Genome-wide analysis of histone modifications that underlie the dynamic changes in gene expression during decidualization in human endometrial stromal cells.

Human endometrial stromal cells (hESCs) undergo a differentiation process with dramatic changes in cell functions during the menstrual cycle, which is called decidualization. This is an important event for implantation of the embryo and successful pregnancy. Defective decidualization can cause implantation failure, miscarriage, and unexplained infertility. A number of genes are up-regulated or down-regulated during decidualization. Recent studies have shown that epigenetic mechanisms are involved in the regulation of decidualization-related genes and that histone modifications occur throughout the genome during decidualization. The present review focuses on the involvement of genome-wide histone modifications in dramatic changes in gene expression during decidualization. The main histone modifications are the increases of H3K27ac and H3K4me3, which activate transcription. C/EBPβ works as a pioneer factor throughout the genome by recruiting p300. This is the main cause of the genome-wide acetylation of H3K27 during decidualization. Histone modifications were observed in both the proximal promoter and distal enhancer regions. Genome editing experiments show that the distal regions have transcriptional activities, which suggests that decidualization induces the interactions between proximal promoter and distal enhancer regions. Taken together, these findings show that gene regulation during decidualization is closely associated with genome-wide changes of histone modifications. This review provides new insights regarding the cases of implantation failure in terms of decidualization insufficiency owing to epigenetic dysregulation, and may lead to novel treatment options for women with implantation failure.

Abstract A057: Microscale analysis of histone modifications in rare cell populations in prostate cancer

Abstract Pre-clinical models have identified diverse epigenetic alterations in prostate cancer that associate with treatment resistance and may also be therapeutic targets. However, these assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input primary tumor samples. Exclusive Liquid Repellant (ELR) platforms pass magnetic bead bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of prostate cancer cell lines with LNCaP, V16D, 49F, 42D, and H660s with serial dilutions was performed to evaluate the sensitivity and specificity of this integrated assay. The CUT&Tag assay was performed in standard fashion on a serial dilution of 50-100,000 cells from LNCaP and V16D calls and showed dropout of recoverable DNA at 50 cells. Sequencing of DNA at these dilutions confirmed identification of differential histone modifications consistent with previously published data. CUT&Tag was adapted to ELR through the serial patterning of each buffer in distinct wells connected by an oil interface. In order to optimize the assay for reproducibility and increased sensitivity, we optimized antibody binding/washing to minimize the protein A-Transposase inhibition. This optimization showed increased antibody binding by 36% compared to protocol defined concentrations. We have now successfully performed this assay with a sensitivity of ~100 cells with recoverable DNA for sequencing to identify heterogenous histone patterns in each cell line. CUT&Tag has improved sensitivity to detect epigenetic alterations that associate with aggressive prostate cancer. The integration of CUT&Tag with ELR improves both sensitivity and reproducibility for epigenetic analysis at low input cell numbers. This assay is now being utilized to analyze patient tumor samples and prostate cancer heterogeneity. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Duane S. Juang, Cole S. Gilsdorf, Shuang G. Zhao, David J. Beebe, Joshua M. Lang. Microscale analysis of histone modifications in rare cell populations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A057.

Pyruvate metabolism controls chromatin remodeling during CD4+ T cell activation

Upon antigen-specific Tcell receptor (TCR) engagement, human CD4+ Tcells proliferate and differentiate, a process associated with rapid transcriptional changes and metabolic reprogramming. Here, we show that the generation of extramitochondrial pyruvate is an important step for acetyl-CoA production and subsequent H3K27ac-mediated remodeling of histone acetylation. Histone modification, transcriptomic, and carbon tracing analyses of pyruvate dehydrogenase (PDH)-deficient Tcells show PDH-dependent acetyl-CoA generation as a rate-limiting step during T activation. Furthermore, Tcell activation results in the nuclear translocation of PDH and its association with both the p300 acetyltransferase and histone H3K27ac. These data support the tight integration of metabolic and histone-modifying enzymes, allowing metabolic reprogramming to fuel CD4+ Tcell activation. Targeting this pathway may provide a therapeutic approach to specifically regulate antigen-driven Tcell activation.

A single-cell multiomic analysis of kidney organoid differentiation

Kidney organoids differentiated from pluripotent stem cells are powerful models of kidney development and disease but are characterized by cell immaturity and off-target cell fates. Comparing the cell-specific gene regulatory landscape during organoid differentiation with human adult kidney can serve to benchmark progress in differentiation at the epigenome and transcriptome level for individual organoid cell types. Using single-cell multiome and histone modification analysis, we report more broadly open chromatin in organoid cell types compared to the human adult kidney. We infer enhancer dynamics by cis-coaccessibility analysis and validate an enhancer driving transcription of HNF1B by CRISPR interference both in cultured proximal tubule cells and also during organoid differentiation. Our approach provides an experimental framework to judge the cell-specific maturation state of human kidney organoids and shows that kidney organoids can be used to validate individual gene regulatory networks that regulate differentiation.

Abstract 4718: Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay

Abstract Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, CUT&RUN requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST CUT&RUN Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the CUT&RUN experiment. Citation Format: Angela H. Guo, Christopher R. Comeau, Fang Chen, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay. [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 4718.