Activation Of Gene Transcription Research Articles

MMP-9-dependent proteolysis of the histone H3 N-terminal tail: a critical epigenetic step in driving oncogenic transcription and colon tumorigenesis.

Matrix metalloproteinase 9 (MMP-9) is a member of the MMP family and has been recently identified as a nuclear protease capable of clipping histone H3 N-terminal tails (H3NT). This MMP-9-dependent H3NT proteolysis is critical for establishing an active state of gene transcription during osteoclast differentiation and melanoma development. However, whether H3NT cleavage by MMP-9 plays a similar role in other cellular events has not been explored. Here, we dissect the functional contribution of MMP-9-dependent H3NT clipping to colonic tumorigenesis by using a combination of genome-wide transcriptome data, ChIP/ChIPac-qPCR, CRISPR/dCas9 gene-targeting system, and in vivo xenograft models. We show that MMP-9 is overexpressed in colon cancer cells and catalyzes H3NT proteolysis to drive transcriptional activation of growth stimulatory genes. Our studies using knockdown and inhibition approaches clearly indicate that MMP-9 mediates transcriptional activation and promotes colonic tumorigenesis in a manner dependent on its protease activity toward H3NT. Remarkably, artificial H3NT proteolysis at target gene promoters with dCAS9-MMP-9 is sufficient for establishing their transcriptional competence in colon cancer cells, underscoring the importance of MMP-9-dependent H3NT proteolysis per se in the transactivation process. Our data establish new functions and mechanisms for MMP-9 in driving the oncogenic transcription program in colon cancer through H3NT proteolysis, and demonstrate how this epigenetic pathway can be exploited as a potential therapeutic target for cancer treatment.

Endocrine Resistance in Breast Cancer: The Role of mTOR Signaling in Mediating Resistance to Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators (SERMs) have been widely prescribed and effective as a first-line endocrine therapy to treat ER+ breast cancer. Tamoxifen, the most used SERM in the treatment of breast cancer, has been shown to be effectively anti-proliferative in breast tissue and has made a tremendous contribution to reducing breast cancer mortality. Vast experimental evidence from seven sources supports tamoxifen’s ability in repressing the expression of estrogen-responsive genes involved in cancer growth. The binding of tamoxifen to the estrogen receptor prevents the recruitment of coactivators to the complex and instead promotes the recruitment of corepressors and histone deacetylases, thus inhibiting transcriptional activation of target genes. However, the issue of endocrine resistance remains a predominant problem with this therapy. Sources have found that endocrine resistance can arise due to dysregulations in the mTOR signaling pathway. Experiments have revealed some hope regarding a mechanism by which we can re-sensitize the breast cancer cells to the therapy, notably by knocking down YAP/TAZ or PSAT1 in the mTOR pathway. Despite this discovery, endocrine resistance prevails due to irregularities in additional pathways. Therefore, subsequent research is crucial to identify more targets that, when knocked down, enable re-sensitization of resistant cells, restoring full therapeutic ability of SERMs in afflicted women.

The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.

Abstract 7558: Novel liquid biopsy based determination of lung cancer histology using a comprehensive epigenomic platform reveals enhancer activity as a key determinant for accurate classification

Abstract BACKGROUND: Accurate molecular characterization of lung cancer including histology classification and therapeutic target profiling is critical for treatment planning and resistance monitoring. For example, detecting lineage transformation from lung adenocarcinoma (LUAD) to small cell lung carcinoma (SCLC) following treatment, an established resistance mechanism, is challenging due to deteriorating patient health and complicated invasive procedures. Here, we describe an accurate lung cancer histological classification using an innovative, multimodal epigenomic liquid biopsy method that comprehensively profiles tumor-specific transcription activation from 1 mL of patient plasma. METHODS: 37 LUAD, 34 SCLC patient plasma samples collected from commercial biobanks, and reference cell lines were profiled for enhancer, promoter and DNA methylation activity to identify epigenomic loci characteristic of LUAD and SCLC. The resulting map of select quantified loci was used as input to a machine learning algorithm that distinguishes SCLC from LUAD, in a cross validated training and testing approach. RESULTS: The epigenomic maps derived from lung cancer patient plasma were highly informative of the regulatory programming of the tumor cells. The enhancers and promoters most strongly linked to lung cancer profiles across the patient population include key, histology-specific driver genes such as NKX2-1, FOXA1, FOXA2, ONECUT2, DLL3, and ASCL1. The resulting classifier was able to accurately predict histology with a cross validated AUC greater than 0.94. In silico simulation results suggest that accurate classification can be achieved with ctDNA content below 1%. Further correlative examination of the SCLC enhancer landscape revealed a partitioning of SCLC patients into molecular subtypes characterized by active enhancer marks at subtype-specific transcripts, including ASCL1, POU2F3, NEUROD1, and YAP1. The transcriptional regulation of ADC targets is apparent through comprehensive epigenomic profiling of plasma in both SCLC and LUAD, with the expression of Claudin 18.2, SEZ6, and others showing a strong dynamic range independent of ctDNA levels. This represents a novel liquid biopsy approach to distinguish LUAD from SCLC and provide simultaneous insight into expression of key targets at clinically relevant ctDNA levels. CONCLUSION: These data demonstrate the feasibility of epigenomic mapping from 1mL of plasma in lung cancer patients to distinguish lung cancer histology. The resulting data is biologically interpretable through the lens of transcriptional activation of key histology specific genes. This liquid-biopsy based approach may provide a non-invasive alternative to invasive and challenging tissue biopsies for biomarker investigation and therapeutic decision making. Citation Format: Jamey Guess, Aparna Gorthi, Anthony D'ippolity, Jonathan Beagan, Travis Clark, Michael Coyne, Tyrone Tamakloe, Baovy Tran, Hyun-Hwan Jeong, Kristian Cibulskis, Hathairat Sawaengsri, Juliann Chmielecki, Corrie A. Painter, J Carl Barrett, Matthew Eaton. Novel liquid biopsy based determination of lung cancer histology using a comprehensive epigenomic platform reveals enhancer activity as a key determinant for accurate classification [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 7558.

Abstract 2067: QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators

Abstract Integrative analyses of CRISPR/Cas9 screening datasets from different sources revealed - besides known oncogenes - a series of transcriptional master regulators as most prominent context-specific dependencies in cancer that remained largely inaccessible to conventional drug discovery approaches. New chemical entities (NCEs) that specifically interfere with a transcription factor (TF) at any level should induce and be detectable based on selective effects on their primary biochemical activity - i.e., the transcriptional activation of specific target genes. However, the definition of direct targets of a TF and, more generally, the unbiased detection of direct transcriptional effects has remained challenging due to the limited time resolution of conventional techniques for gene perturbation and transcriptional profiling. To overcome this limitation, QUANTRO Therapeutics relies on SLAMseq technology that, by metabolic labeling de novo-synthesized RNA immediately after intervention and conserving the information for later readout, provides unprecedent time-resolution in transcriptomic analysis. Aiming to target previously undruggable targets, we set out to first build definitive knowledge around their direct effect on transcription, inducing their quick protein degradation through the auxin-inducible degron (AID) technology followed by immediate transcriptional readout though SLAMseq -before any secondary, confounding effect could appear in the cell. The targets´ mRNA fingerprints produced are then used as a reference and cross-compared with existing and new chemical entities to: (i) benchmark the real mode of action of existing drugs; (ii) identify new drug candidates that specifically interfere with disease-causing transcriptional programs in cancer; (iii) interrogate the efficiency, selectivity, and potency of drug candidates during Hit-validation and Hit-to-Lead activities. Proof of concept data demonstrated that time-resolved transcriptional profiling can reveal drugs´ mode of action with unprecedent precision and sensitivity, when drugs act on Targets that have a direct effect on transcriptional regulation - such as TFs and signaling pathways. Building on this knowledge, QUANTRO Therapeutics unleashed the unique potential of mRNA fingerprinting technology for HTS-compatible drug discovery by (1) establishing scalable and automated workflows for metabolic RNA labeling, treatment of cells, and RNA extraction; (2) developing algorithms for combinatorial mRNA fingerprint-readout that allow for the simultaneous assessment of TF signatures by multiplexed targeted RNA sequencing (QUANTROseq), thus transforming the precision and scope of cell-based compound screens. Citation Format: Arianna Sabò, Nina Fasching Griesmayr, Tobias Neumann, Raphael Manzenreither, Maja Ivankovic, Rodrigo Pacheco Valamatos Costa, Sarah Rieser, Sara Scinicariello, Petr Triska, Anna Stingeder, Astrid Gruss, Ryan Cubero, Olga Frank, Ivica Sowemimo, Paul Kirchgatterer, Adriana Cantoran, Stefan Ameres, Johannes Zuber, Michael Bauer. QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators [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 2067.

Abstract 3037: Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion

Abstract Unveiling the molecular mechanisms underlying sex-specific differences in cancer initiation, progression and treatment outcomes will provide novel insights that will advance cancer research and clinical care. This study highlights an unexpected, sex-biased role of the oncogene Yap1 in SHH medulloblastoma (MB). We discovered that Yap1 deletion in SmoM2-driven SHH MB significantly prolongs survival in male but not female mice. Using an integrated multi-omics approach, we show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation genes such as NeuroD1 and Zic1/2. Interestingly, while Yap1 is essential for maintaining cancer stem cells in both sexes, it plays a more critical role in immune evasion in males. Specifically, YAP1 regulates the expression of an immune checkpoint molecule Cd276 to suppress T cell function. Blocking CD276 or deleting Yap1 is sufficient to significantly reverse T cell suppression in males but not females. Furthermore, YAP1 direct targets of transcriptional regulation, including CD276, predicts survival in male but not female patients across multiple human cancers, suggesting that our findings have broader implications beyond medulloblastomas and is conserved across species. This study provides compelling evidence for male-biased susceptibility to Yap1 inhibition and uncovers the YAP1-CD276 axis as a sexually diverse pathway in T cell suppression in tumors. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Han Nhat Tran, Jose Maldonado, Rachael McMinimy, Hannah Borland, Shu-hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-specific differences in Yap1 and Cd276 function and expression regulate medulloblastoma progression and immune evasion [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 3037.

Abstract 108: Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model

Abstract Mutations in STK11 that lead to loss of its protein LKB1 occur in 15-20% of non-squamous non-small cell lung cancer (NSCLC) and are frequently co-mutated with oncogenic KRAS mutations. KRAS and STK11 co-mutant NSCLC is associated with poor prognosis, low PD-L1 expression, low T-cell infiltration, and poor response to immune-checkpoint inhibitors (ICI). Loss of LKB1 leads to altered transcriptional programs with the activation of CREB-dependent gene transcription and changes in the tumor microenvironment including recruitment of suppressive myeloid cells. We used the syngeneic Kras mutant murine CMT167 tumor cell line that is wild-type for Tp53 and Stk11 and sensitive to ICI and generated isogeneic Stk11 knockout cell lines (CMT167-Stk11-KO) by CRISPR/Cas-9 deletion. Loss of Stk11 recapitulated LKB1-loss transcriptional signatures and rendered CMT167 tumors resistant to ICI. Single-cell RNA sequencing (10x) of implanted tumors demonstrated significantly less CD8 T-cells and increased neutrophils (PMN) in Stk11-KO versus Stk11-WT tumors. Analysis of differentially expressed genes in the tumor compartment identified increased expression of complement pathway genes including the central mediator C3 in CMT-167-Stk11-KO tumors. Examination of a well characterized clinical cohort of human KRAS-mutant NSCLC patient samples with and without STK11 mutation confirmed the increased expression of complement C3 in STK11-mutant tumors. Analysis of a human NSCLC cell line panel demonstrated that C3 expression was modulated by LKB1. Knockout of C3 in CMT167-Stk11-KO tumor resulted in dramatic inhibition of tumor growth and re-sensitized CMT167-Stk11-KO tumors to ICI treatment in WT mice. Using C3-/- knockout mice, this effect was determined to be reliant on tumor-derived C3 and dependent on adaptive immunity as no difference in growth of CMT167-Stk11-KO tumors occurred in nude mice or in WT mice after CD8 T-cell depletion. We then compared the transcriptome of Stk11-KO tumor cells with and without C3 deletion, and observed that C3 upregulates the expression of Cxcl1, Cxcl2 and Cxcl3 which mediate PMN recruitment and activation. Since these chemokines ligate Cxcr2, we asked whether Cxcr2 mediated tumor growth and ICI resistance in Stk11-KO tumors. While treatment with single-agent Cxcr2 inhibitor and anti-PD-1 therapy had no effect on tumor growth, the combination resulted in significant suppression of tumor growth in vivo. These results support a role for tumor-derived C3 suppressing CD8 T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in patients with STK11-mutant NSCLC. Citation Format: Sora Suzuki, Catrina Ting, Bojidar Kandar, Te-An Chen, Anm Nazmul Khan, Thejaswini Giridharan, Brahm Segal, Edwin H. Yau. Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model [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 108.

Abstract 6258: Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA

Abstract Cell-free DNA (cfDNA) circulating in blood plasma arises primarily from cellular chromatin fragmentation and release due to cell death. Studies have demonstrated that the assessment of fragmentomic features of cfDNA enables gene expression inference and tissue-of-origin classification with potential applications for noninvasive cancer detection. However, due to low depth of coverage of sites of interest, current whole genome sequencing (WGS) methods have not been able to infer expression of individual genes or limited gene sets. Therefore, we developed a targeted sequencing approach that enables individual gene expression inference from cfDNA fragmentation patterns based on transcriptional start-site gene activation probability (TSS-GAP). To assess the sensitivity of this method for cfDNA-inferred gene expression profiling, we applied TSS-GAP to blends of micrococcal nuclease (MNase)-digested DNA from a cancer cell line (LS180) and sorted peripheral blood mononuclear cells (PBMC), with LS180 DNA at varying concentrations (0.1-10%). This enabled the determination of the limit of detection (LoD), relative to healthy donor background levels, on the basis of the genes’ activation probabilities (TSS-GAP scores). We found 936 genes with high inferred expression in the LS180 cancer cell line and low inferred expression in non-cancer samples (i.e. CD4, CD14, and plasma cfDNA from 4 healthy donors) and used those for healthy donor background and LoD assessment. Using these gene-level background values as cutoffs, blends were assessed to determine the lowest blend level at which expression of the gene was able to be differentiated from background. Out of the 936 genes, 326 (34.8%) were detectable at a blend level of 0.1%, 59 (6.3%) at 0.3%, 31 (3.3%) at 1%, 19 (2.0%) at 3%, and 39 (4.2%) at 10%. In total, 416 genes could be detected by TSS-GAP at levels 1% of or lower above background, highlighting the potential of our method for detecting expression of certain genes even from a small fraction of cells contributing cfDNA to liquid biopsy samples. Further expansion of this work could consider the utility of such a method for circulating tumor DNA (ctDNA) detection, cfDNA tissue-of-origin classification, and deconvolution of cell types contributing to cfDNA found in blood. Citation Format: Emily Leff, Alexander Tseng, Victoria Cheung, Ehsan Tabari, Kimberly Walter, Alex Lovejoy, Cheng-Ho J. Lin. Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA [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 6258.

H3K4 methylation regulates development, DNA repair, and virulence in Mucorales

Mucorales are basal fungi that opportunistically cause a potentially fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic fungi. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis.

Unveiling Alterations of Epigenetic Modifications and Chromatin Architecture Leading to Lipid Metabolic Reprogramming during the Evolutionary Trastuzumab Adaptation of HER2-Positive Breast Cancer.

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

Role of reactive oxygen species (ROS) signaling pathway in the wound-induced spore formation of Pyropia yezoensis

Asexual spore formation of Pyropia yezoensis significantly impacts yield. After mechanical sectioning of leafy thalli into fragments, somatic cells rapidly develop into a large number of wound-induced spores (WIS, a form of asexual spores). Recent studies on P. yezoensis have demonstrated a notable increase in reactive oxygen species (ROS) during this process; however, the regulatory mechanism underlying this ROS signaling remains largely unclear. This paper shows that in P. yezoensis, ROS signals play a crucial role in WIS formation. When treated with the ROS-producing enzyme NADPH oxidase inhibitor diphenyleneiodonium chloride, P. yezoensis thalli fragments strongly repressed WIS formation, accompanied by a significant decrease of ROS levels, malondialdehyde contents, and activities of NADPH oxidase and antioxidant enzymes. The expression levels of respiratory burst oxidase homolog genes, aquaporin protein genes, superoxide dismutase genes, glutathione S-transferase genes, and glutathioneperoxidase genes were also decreased, thus inhibiting WIS formation. As an upstream signal, ROS promote ion exchange and metabolism both inside and outside of cells within the first 12 h of wound stress. Following 12 h of wound stress, transcriptional activation of specific genes associated with cell cycle progression—which promotes cell division and dedifferentiation under normal conditions—was also blocked by diphenyleneiodonium chloride. In addition, the generated ROS signals also regulated the expression of mitogen-activated protein kinase family genes under wound stress. Overall, this study provides novel insights into the molecular mechanism underlying the ROS signaling regulation of WIS formation in P. yezoensis.

Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass

BackgroundSwitchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress.ResultsIn total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls.ConclusionOur study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.

Targeting of mitochondrial fission through natural flavanones elicits anti-myeloma activity.

Mitochondrial alterations, often dependent on unbalanced mitochondrial dynamics, feature in the pathobiology of human cancers, including multiple myeloma (MM). Flavanones are natural flavonoids endowed with mitochondrial targeting activities. Herein, we investigated the capability of Hesperetin (Hes) and Naringenin (Nar), two aglycones of Hesperidin and Naringin flavanone glycosides, to selectively target Drp1, a pivotal regulator of mitochondrial dynamics, prompting anti-MM activity. Molecular docking analyses were performed on the crystallographic structure of Dynamin-1-like protein (Drp1), using Hes and Nar molecular structures. Cell viability and apoptosis were assessed in MM cell lines, or in co-culture systems with primary bone marrow stromal cells, using Cell Titer Glo and Annexin V-7AAD staining, respectively; clonogenicity was determined using methylcellulose colony assays. Transcriptomic analyses were carried out using the Ion AmpliSeq™ platform; mRNA and protein expression levels were determined by quantitative RT-PCR and western blotting, respectively. Mitochondrial architecture was assessed by transmission electron microscopy. Real time measurement of oxygen consumption was performed by high resolution respirometry in living cells. In vivo anti-tumor activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. Hes and Nar were found to accommodate within the GTPase binding site of Drp1, and to inhibit Drp1 expression and activity, leading to hyperfused mitochondria with reduced OXPHOS. In vitro, Hes and Nar reduced MM clonogenicity and viability, even in the presence of patient-derived bone marrow stromal cells, triggering ER stress and apoptosis. Interestingly, Hes and Nar rewired MM cell metabolism through the down-regulation of master transcriptional activators (SREBF-1, c-MYC) of lipogenesis genes. An extract of Tacle, a Citrus variety rich in Hesperidin and Naringin, was capable to recapitulate the phenotypic and molecular perturbations of each flavanone, triggering anti-MM activity in vivo. Hes and Nar inhibit proliferation, rewire the metabolism and induce apoptosis of MM cells via antagonism of the mitochondrial fission driver Drp1. These results provide a framework for the development of natural anti-MM therapeutics targeting aberrant mitochondrial dependencies.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.

The BAS chromatin remodeler determines brassinosteroid-induced transcriptional activation and plant growth in Arabidopsis

Brassinosteroid (BR) signaling leads to the nuclear accumulation of the BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor, which plays dual roles in activating or repressing the expression of thousands of genes. BZR1 represses gene expression by recruiting histone deacetylases, but how it activates transcription of BR-induced genes remains unclear. Here, we show that BR reshapes the genome-wide chromatin accessibility landscape, increasing the accessibility of BR-induced genes and reducing the accessibility of BR-repressed genes in Arabidopsis. BZR1 physically interacts with the BRAHMA-associated SWI/SNF (BAS)-chromatin-remodeling complex on the genome and selectively recruits the BAS complex to BR-activated genes. Depletion of BAS abrogates the capacities of BZR1 to increase chromatin accessibility, activate gene expression, and promote cell elongation without affecting BZR1's ability to reduce chromatin accessibility and expression of BR-repressed genes. Together, these data identify that BZR1 recruits the BAS complex to open chromatin and to mediate BR-induced transcriptional activation of growth-promoting genes.

A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and invivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

Anti-obesity effect of butyrate links to modulation of gut microbiome and epigenetic regulation of muscular circadian clock

The role of the muscle circadian clock in regulating oxidative metabolism exerts a significant influence on whole-body energy metabolism; however, research on the connection between the muscle circadian clock and obesity is limited. Moreover, there is a lack of studies demonstrating the regulatory effects of dietary butyrate on muscle circadian clock and the resulting antiobesity effects. This study aimed to investigate the impacts of dietary butyrate on metabolic and microbiome alterations and muscle circadian clock in a diet-induced obesity model. Male Sprague–Dawley rats were fed a high-fat diet with or without butyrate. Gut microbiota and serum metabolome were analyzed, and molecular changes were examined using tissues and a cell line. Further correlation analysis was performed on butyrate-induced results. Butyrate supplementation reduced weight gain, even with increased food intake. Gut microbiome analysis revealed an increased abundance of Firmicutes in butyrate group. Serum metabolite profile in butyrate group exhibited reduced amino acid and increased fatty acid content. Muscle circadian clock genes were upregulated, resulting in increased transcription of fatty acid oxidation-related genes. In myoblast cells, butyrate also enhanced pan-histone acetylation via histone deacetylase inhibition, particularly modulating acetylation at the promoter of circadian clock genes. Correlation analysis revealed potential links between Firmicutes phylum, including certain genera within it, and butyrate-induced molecular changes in muscle as well as phenotypic alterations. The butyrate-driven effects on diet-induced obesity were associated with alterations in gut microbiota and a muscle-specific increase in histone acetylation, leading to the transcriptional activation of circadian clock genes and their controlled genes.

Abstract B009: TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in Triple-Negative Breast Cancer

Abstract Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from a lack of targeted treatment options. There is a high priority to identify effective molecular markers or driver factors specific to TNBC patients for targeted therapies. The Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/Fibroblast growth factor-inducible 14 (Fn14) pathway is one such signalling cascade implicated in the pathogenesis of TNBC and other solid tumours. However, the clinical significance and biological roles of deregulated TWEAK/Fn14 signalling in regulating the epigenetic plasticity and transcriptome of TNBCs remain elusive. In this study, we performed integrative transcriptomic and epigenomic analyses of TNBC cell lines and patient samples to characterise the gene regulatory roles of TWEAK/Fn14 pathway in remodelling the super-enhancer (SE) landscape of TNBC tumours. Here, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of NAMPT, which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients. Citation Format: Yinghui Li, Nicholas Sim, Jean-Michel Carter, Kamalakshi Deka, Benita Kiat Tee Tan, Yirong Sim, Suet-Mien Tan. TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B009.

RUNX1/NPM1/H3K4me3 complex contributes to extracellular matrix remodeling via enhancing FOSL2 transcriptional activation in glioblastoma

Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1’s transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia

BackgroundVascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease.MethodsWire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype.ResultsPhenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation.ConclusionsThe present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.Graphical abstract