Chromatin-associated Proteins Research Articles

HIRA and dPCIF1 coordinately establish totipotent chromatin and control orderly ZGA in Drosophila embryos

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA.

A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization.

A Plasmodium falciparum MORC protein complex modulates epigenetic control of gene expression through interaction with heterochromatin.

Dynamic control of gene expression is critical for blood stage development of malaria parasites. Here, we used multi-omic analyses to investigate transcriptional regulation by the chromatin-associated microrchidia protein, MORC, during asexual blood stage development of the human malaria parasite Plasmodium falciparum. We show that PfMORC (PF3D7_1468100) interacts with a suite of nuclear proteins, including APETALA2 (ApiAP2) transcription factors (PfAP2-G5, PfAP2-O5, PfAP2-I, PF3D7_0420300, PF3D7_0613800, PF3D7_1107800, and PF3D7_1239200), a DNA helicase DS60 (PF3D7_1227100), and other chromatin remodelers (PfCHD1 and PfEELM2). Transcriptomic analysis of PfMORCHA-glmS knockdown parasites revealed 163 differentially expressed genes belonging to hypervariable multigene families, along with upregulation of genes mostly involved in host cell invasion. In vivo genome-wide chromatin occupancy analysis during both trophozoite and schizont stages of development demonstrates that PfMORC is recruited to repressed, multigene families, including the var genes in subtelomeric chromosomal regions. Collectively, we find that PfMORC is found in chromatin complexes that play a role in the epigenetic control of asexual blood stage transcriptional regulation and chromatin organization.

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

Abstract Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

The role of HMGB2 in the immune response of Nile tilapia (Oreochromis niloticus) to streptococcal infection

High mobility group protein B2 (HMGB2) is an abundant chromatin-associated protein with pivotal roles in transcription, cell proliferation, differentiation, inflammation, and tumorigenesis. However, its immune function in Nile tilapia (Oreochromis niloticus) remains unclear. In this study, we identified a homologue of HMGB2 from Nile tilapia (On-HMGB2) and investigated its functions in the immune response against streptococcus infection. The open reading frame (ORF) of On-HMGB2 spans 642 bp, encoding 213 amino acids, and contains two conserved HMG domains. On-HMGB2 shares over 80 % homology with other fish species and 74%–76 % homology with mammals. On-HMGB2 was widely distributed in various tissues, with its highest transcript levels in the liver and the lowest in the intestine. Knockdown of On-HMGB2 promoted the inflammatory response in Nile tilapia, increased the bacterial load in the tissues, and led to elevated mortality in Nile tilapia following Streptococcus agalactiae infection. Taken together, On-HMGB2 significantly influences the immune system of Nile tilapia in response to streptococcus infection.

Decoding the genomic landscape of chromatin-associated biomolecular condensates

Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.

Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision.

Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack of cofactor-dependent active sites of transcription factors further complicate the quantitative analysis of these critical processes. Here, we create a genetically encoded label-free sensor to identify the interaction between a motif of the MYC transcription factor, a primary cancer driver, and WDR5, a chromatin-associated protein hub. Using an engineered nanopore equipped with this motif, WDR5 is probed through reversible captures and releases in a one-by-one and time-resolved fashion. Our single-molecule kinetic measurements indicate a weak-affinity interaction arising from a relatively slow complex association and a fast dissociation of WDR5 from the tethered motif. Further, we validate this subtle interaction by determinations in an ensemble using single nanodisc-wrapped nanopores immobilized on a biolayer interferometry sensor. This study also provides the proof-of-concept for a sensor that reveals unique recognition signatures of different protein binding sites. Our foundational work may be further developed to produce sensing elements for analytical proteomics and cancer nanomedicine.

Initial Characterization of WDR5B Reveals a Role in the Proliferation of Retinal Pigment Epithelial Cells.

The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

Cohesin composition and dosage independently affect early development in zebrafish.

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

Abstract B005: Regulation of replication-induced PARP1/PARP2 activation by base excision repair: Implications for PARP and PARG inhibitor resistance

Abstract Acquired poly (ADP-ribose) polymerases (PARP)-inhibitor (PARPi) and/or poly (ADP-ribose) glycohydrolase (PARG)-inhibitor (PARGi) resistance in BRCA1/2-defective tumors is common, emphasizing the need for identifying new targets. Protein poly ADP-ribosylation (PARylation) is a posttranslational modification of proteins catalyzed by PARP to form poly-(APD-ribose) (PAR) chains on itself and chromatin-associated proteins in response to DNA damage. Our study reveals that PARylation occurs in a replication-, PARP1-, and PARP2-dependent manner even in undamaged cells. Using proximity labeling, specifically Split-TurboID, we found that replication-dependent PARylation recruits the base excision repair (BER) factors XRCC1, POLB, APTX, and LIG3. These factors, in turn, suppress further PARylation. The BER factors XRCC1, POLB, APTX, or LIG3 promote PARPi and PARGi resistance as loss of these replication-associated BER (R-BER) factors re-sensitizes cancer cells to these inhibitors. Of interest, BRCA1 and BRCA2 localize with R-BER factors in undamaged cells. Lastly, XRCC1, POLB, APTX, and LIG3 depletion activate the S-phase checkpoint kinase CHK1, and targeting either this kinase or ATR under these conditions overcomes PARGi resistance in glioblastoma and ovarian cancer cells. In conclusion, our data highlight the crucial role of R-BER in protecting the cell during S-phase and propose a potential intervention strategy for resistant tumors. Citation Format: Md Ibrahim, Md Maruf Khan, Wynand P. Roos, Rasha Q. Al-Rahaleh, Faisal Hayat, Marie E. Migaud, Robert W. Sobol. Regulation of replication-induced PARP1/PARP2 activation by base excision repair: Implications for PARP and PARG inhibitor resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B005.

Abstract PR012: KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer

Abstract The estrogen receptor (ER) is expressed in ∼70% of breast cancers where it functions as a critical transcription factor that regulates cell growth and tumor progression. ER inhibition is a mainstay treatment for ER-positive (ER+) breast cancer, but despite the clinical benefit of targeting ER signaling, breast cancer remains the second leading cause of cancer-related death in women, with ∼50% of deaths resulting from ER+ tumors. While recurrent and metastatic ER+ tumors are typically resistant to endocrine therapies they continue to rely on ER signaling suggesting that disruption of other chromatin associated complexes that mediate ER-driven gene expression may present an alternative therapeutic strategy. One chromatin modifier important in regulating ER function is the histone acetyltransferase KAT6A. KAT6A is amplified and/or overexpressed in ∼15% of ER+ tumors and a novel KAT6A/B inhibitor has entered clinical trials for advanced breast cancer. However, as with most single agent therapies, compensatory mechanisms may attenuate the effects of KAT6A/B inhibition on ER-driven gene expression. We hypothesized that the dual targeting of distinct chromatin complexes would enhance the effects of KAT6A/B inhibition, by altering chromatin state and transcriptional output. To that end we performed an epigenetic focused CRISPR-Cas9-based functional genetic screen to identify chromatin-associated proteins that enhance KAT6A/B inhibition. CRISPR guides targeting MEN1 (encoding Menin) were among our top hits as sensitizers to treatment with the KAT6A/B inhibitor PF-9363. We confirmed that co-treatment of PF-9363, and the Menin inhibitor, SNDX-5613, had strong synergistic anti-proliferative effects in a panel of ER+ breast cancer cell lines. Mechanistically, our data revealed that KAT6A/B and Menin cooperatively regulate ER-driven gene expression and chromatin accessibility, both through direct effects on ESR1 expression as well as through effects at ER target genes. Specifically, we found that KAT6A and Menin-KMT2A complexes co-localized at promoters of ER target genes and treatment with KAT6A/B or Menin inhibitors displaced KAT6A and Menin/KMT2A from ER target genes, respectively. Yet only the combination effectively displaced both complexes from chromatin, which produced more dramatic changes in ER-directed gene expression than either small molecule alone. Combined displacement of KAT6A and Menin/KMT2A at ER-regulated genes induced loss of an active chromatin landscape characterized by reduction of activating histone modifications and loss of RNA Polymerase II binding to ER target loci. Importantly, the combination of KAT6A/B and Menin inhibition was effective in a panel of ER+ patient-derived organoid models as well as in various models of endocrine resistance, including models with ESR1 mutations, NF1 loss, and FOXA1 mutations. Both KAT6A/B and Menin inhibitors are in Phase I/II clinical trials and have shown manageable toxicity profiles to date. This combination represents a novel potential therapeutic combination for ER+ breast cancer. Citation Format: Sarah Naomi Olsen, Bryn Anderson, Charles Hatton, Rinath Jeselsohn, Myles Brown, Eneda Toska, Scott Armstrong. KAT6A/B and Menin-MLL complexes coordinately regulate estrogen receptor-driven gene expression programs in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR012.

Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Disrupting the interaction between a p53 gain-of-function mutant and the transcriptional co-activator PC4 reverses drug resistance in cancer cells.

PC4 is a chromatin-associated protein and transcriptional coactivator whose role in gene regulation by wild-type p53 is now well known. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. We show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. Here, we introduced a peptide that disrupts the PC4-R273Hp53 interaction to tumor cells bearing the R273HTP53 gene, which led to a lowering of MDR1 expression and abrogation of drug resistance in a mutant-specific manner. The results suggest that the PC4-R273Hp53 interaction may be a promising target for reducing proliferation and drug resistance in tumors.

Prediction of cell-type-specific cohesin-mediated chromatin loops based on chromatin state

Chromatin loop is of crucial importance for the regulation of gene transcription. Cohesin is a type of chromatin-associated protein that mediates the interaction of chromatin through the loop extrusion. Cohesin-mediated chromatin interactions have strong cell-type specificity, posing a challenge for predicting chromatin loops. Existing computational methods perform poorly in predicting cell-type-specific chromatin loops. To address this issue, we propose a random forest model to predict cell-type-specific cohesin-mediated chromatin loops based on chromatin states identified by ChromHMM and the occupancy of related factors. Our results show that chromatin state is responsible for cell-type-specificity of loops. Using only chromatin states as features, the model achieved high accuracy in predicting cell-type-specific loops between two cell types and can be applied to different cell types. Furthermore, when chromatin states are combined with the occurrence frequency of CTCF, RAD21, YY1, and H3K27ac ChIP-seq peaks, more accurate prediction can be achieved. Our feature extraction method provides novel insights into predicting cell-type-specific chromatin loops and reveals the relationship between chromatin state and chromatin loop formation.

Abstract 7026: Multi-omic genomic mapping with long read sequencing

Abstract Gene transcription is regulated by the complex interplay between histone post-translational modifications (PTMs), chromatin associated proteins (CAPs), and DNA methylation (DNAme). Mapping their genomic locations and examining the relationships between these chromatin elements is a powerful approach to decipher mechanisms of disease, thereby enabling discovery of novel biomarkers and therapeutics. Leading epigenomic mapping technologies (e.g., ChIP-seq, CUT&RUN) rely upon DNA fragmentation to isolate regions of interest for sequencing on short read platforms (e.g., Illumina). This strategy leads to substantial loss of contextual information regarding the surrounding DNA, precluding the identification of multiple co-occurring epigenomic features on a single DNA molecule. By contrast, long-read sequencing (LRS) platforms are capable of sequencing very long reads from a single molecule (typically >10kb), allowing relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. Here we report a robust multi-omic method that leverages LRS to simultaneously profile histone PTMs (or CAPs), DNAme, and parental haplotype in a single assay. This nondestructive, epigenomic mapping approach leverages a novel DNA methyltransferase fusion protein (pAG-M.EcoGII) to label DNA underneath antibody-targeted chromatin features, thereby marking sites of interest while preserving DNA molecules intact for LRS. Inspired by our work with state-of-the-art immunotethering-based approaches (CUT&RUN/CUT&Tag), nuclei are bound to magnetic beads to streamline and automate sample processing. Next, adenosines nearby antibody-targeted chromatin features are methylated with pAG-M.EcoGII, which are then directly read from genomic DNA using Oxford Nanopore Technologies or Pacific Biosciences LRS platforms. To determine the capabilities and limitations of this assay, we tested multiple chromatin targets in various cell lines. Importantly, this method is highly reproducible across biological replicates, and highly concordant with orthogonal SRS assays (e.g., CUT&RUN). Further, we showed that this method is a true multi-omic approach by simultaneously profiling histone PTMs, native DNAme (5mC), and parental single-nucleotide variants from single DNA molecules within a single reaction. Finally, this workflow preserves chromatin integrity for LRS, revealing heterogeneity (e.g., haplotype or paternal origin) within/between data types and providing access to previously unmappable genomic regions (e.g., centromeres). Citation Format: Bryan J. Venters, Paul W. Hook, Vishnu S. Kumary, Alli R. Hickman, James T. Anderson, Anup Vaidya, Ryan J. Ezell, Jonathan M. Burg, Zu-Wen Sun, Martis W. Cowles, Winston Timp, Michael-Christopher Keogh. Multi-omic genomic mapping with long read sequencing [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 7026.

Abstract 7027: Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq

Abstract DNA methylation (DNAme) is an epigenetic mark that includes the modification of cytosine resides (5mC) within CpG islands. In addition to well characterized roles regulating gene expression, imprinting and silencing parasitic DNA elements, the misregulation of DNAme is implicated in multiple diseases. Evidence is emerging that DNAme is not an independent epigenetic mark but rather closely linked to the post translational modification (PTM) of histone proteins. However, examining the relationship between 5mC and PTMs are hampered by the usual approach of independent analyses that cannot establish a direct linkage. Furthermore, the traditional approach to measure 5mC relies upon harsh bisulfite chemical conversation of DNA, which introduces damage and systemic biases. To address these limitations, we developed Targeted Enzymatic Methylation-sequencing (TEM-seq): an ultra-sensitive multi-omic genomic mapping technology that delivers high resolution DNAme profiles at epitope-defined chromatin features. Importantly this assay is capable of directly examining the link between 5mC and histone PTMs and/or chromatin associated proteins (ChAPs). The TEM-seq workflow integrates CUT&RUN for genomic mapping with NEB’s enzymatic methyl-seq (EM-seq) for unbiased DNAme analysis. CUT&RUN is a highly sensitive, standards-driven assay that uses antibodies to locally tether protein A/G-micrococcal nuclease (pAG-MNase) to chromatin within intact cells or nuclei, followed by controlled activation of the MNase to cleave nearby DNA. EM-seq then enzymatically converts [5mC/5hmC] to generate single-base resolution, unbiased DNAme profiles from sub-ng samples. To determine the capabilities and limitations of this novel approach, we tested multiple chromatin targets in various cell lines. We demonstrate that TEM-seq is highly reproducible, specific, and efficient (<10% off-target binding, >90% enzymatic conversion of DNAme, <0.5% conversion of unmethylated DNA). TEM-seq is also highly-sensitive, requiring only five million short-read sequences per assay (10-50x less than whole-genome 5mC-sequencing), demonstrating the disruptive potential of the assay to enable cost-effective approach for targeted DNAme analysis. Finally, we leveraged TEM-seq to gain mechanistic insights to Rett syndrome a neurodegenerative disorder that results from mutations in the 5mC-binding domain of the MECP2 gene. Citation Format: Keith E. Maier, Vishnu U. Sunitha Kumary, Bryan J. Venters, Allison Hickman, Anup Vaidya, James Anderson, Ryan Ezell, Jonathan M. Burg, Louise Williams, Chaithanya Ponnaluri, Pierre Esteve, Isaac Meek, Zu-wen Sun, Martis W. Cowles, Sriharsa Pradhan, Michael-Christopher Keogh. Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via TEM-seq [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 7027.

Abstract 5116: Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. We previously reported that ALL cells are vulnerable to metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and we and others have reported it interacts with chromatin-associated proteins to regulate gene expression in response to metabolic stress. To investigate AMPK’s mechanistic role in epigenetic gene regulation under metabolic stress, and identify new AMPK partners, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1. Following treatment with the allosteric AMPK activator PF-06409577 (PF064) for 1h, to mimic the cell’s rapid response to energy/metabolic stress, the samples underwent mass spectrometry-based proteomics. Proteomics data analysis identified novel altered (enhanced or reduced) AMPK protein-protein interactions (PPI) following PF064 treatment. The GO analysis of proteins interacting with AMPK showed that metabolic stress enhances PPIs involved in regulating gene expression, cellular metabolic processes, DNA repair, chromatin organization, histone modifications, etc. More importantly, we identified that AMPK interacted with members of the non-canonical Polycomb repressive complex 1 (PRC1.1) such as PCGF1, RING1, KDM2B, BCORL1, USP7. PRC1 is a known epigenetic regulator that monoubiquitinylates histone H2A at Lys119 (H2AK119ub) to control transcription and regulate gene expression. Using Co-IP, we confirmed these interactions in KASUMI-2 (Bp-ALL) and KE-37 (T-ALL) cells. When cells were treated with PF064 or 991 (another AMPK activator), we found that the interactions of AMPK with the catalytic core of PCR1.1 (PCGF1, RING1) were increased compared to untreated cells. To examine the effect of activated AMPK on PCR1.1 activity, we determined the level of H2AK119ub in ALL cells treated with PF064 and 991 for 1h. We found that the level of H2AK119ub protein expression was increased in ALL-treated cells indicating the PRC1.1 was activated. Together, these data suggest that stress-induced AMPK activation increased the catalytic activity of the PRC1.1 via PPI in ALL. Our findings are consistent with our previous published RNA-Seq and ChIP-Seq data indicating an epigenetic role for AMPK in altering gene expression examined under metabolic stress (two thirds exhibited downregulation), and provide insights into the AMPK-driven mechanism(s) involved. Further elucidation of the uncovered PPIs and its mechanistic role(s) may lead to potential epigenetic-based strategies to overcome resistance/refractoriness in ALL and other malignancies. Citation Format: Anna Shvab, Guy J. Leclerc, Julio C. Barredo. Stress-induced AMPK interactome analysis reveals novel protein-protein interactions associated with chromatin remodeling in acute lymphoblastic leukemia [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 5116.

Increased genomic instability and reshaping of tissue microenvironment underlie oncogenic properties of Arid1a mutations.

Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.