Enhancer Region Research Articles

Energetic particle precipitation influences global secondary ozone distribution

The secondary ozone layer is a global peak in ozone abundance in the upper mesosphere-lower thermosphere (UMLT) around 90-95 km. The effect of energetic particle precipitation (EPP) from geomagnetic processes on this UMLT ozone remains largely unexplored. In this research we investigated how the secondary ozone responds to EPP using satellite observations. In addition, the residual Mean Meridional Circulation (MMC) derived from model simulations and the atomic oxygen [O], atomic hydrogen [H], temperature measurements from satellite observations were used to characterise the residual circulation changes during EPP events. We report regions of secondary ozone enhancement or deficit across low, mid and high latitudes as a result of global circulation and transport changes induced by EPP. The results are supported by a sensitivity test using an empirical model.

ISLS: An Illumination-Aware Sauce-Packet Leakage Segmentation Method.

The segmentation of abnormal regions is vital in smart manufacturing. The blurring sauce-packet leakage segmentation task (BSLST) is designed to distinguish the sauce packet and the leakage's foreground and background at the pixel level. However, the existing segmentation system for detecting sauce-packet leakage on intelligent sensors encounters an issue of imaging blurring caused by uneven illumination. This issue adversely affects segmentation performance, thereby hindering the measurements of leakage area and impeding the automated sauce-packet production. To alleviate this issue, we propose the two-stage illumination-aware sauce-packet leakage segmentation (ISLS) method for intelligent sensors. The ISLS comprises two main stages: illumination-aware region enhancement and leakage region segmentation. In the first stage, YOLO-Fastestv2 is employed to capture the Region of Interest (ROI), which reduces redundancy computations. Additionally, we propose image enhancement to relieve the impact of uneven illumination, enhancing the texture details of the ROI. In the second stage, we propose a novel feature extraction network. Specifically, we propose the multi-scale feature fusion module (MFFM) and the Sequential Self-Attention Mechanism (SSAM) to capture discriminative representations of leakage. The multi-level features are fused by the MFFM with a small number of parameters, which capture leakage semantics at different scales. The SSAM realizes the enhancement of valid features and the suppression of invalid features by the adaptive weighting of spatial and channel dimensions. Furthermore, we generate a self-built dataset of sauce packets, including 606 images with various leakage areas. Comprehensive experiments demonstrate that our ISLS method shows better results than several state-of-the-art methods, with additional performance analyses deployed on intelligent sensors to affirm the effectiveness of our proposed method.

Abstract A013: Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer

Abstract Recent whole-genome sequencing studies have unveiled intriguing hotspot mutations within non-coding genomic regions, yet their implications in cancer remain unknown. Here, we characterized the role of twin hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer: chr8: 29,952,919 G>A and chr8: 29,952,919 C>T. Either mutation is found in ~20% of bladder tumors, and the mutations do not typically co-occur. Through a comprehensive analysis integrating ChiP-seq, ATAC-seq, and Hi-C data from bladder cancer cell lines, we observed that these mutations co-localize with enhancer elements that interact with multiple nearby genes. In vitro dual luciferase reporter assays showed that both mutations are functional and likely affect gene expression in a genomic context. CRISPR-Cas9-mediated knocking out of the putative enhancer region identified LEPROTL1, DCTN6, and SARAF as target genes that are likely co-regulated, which was further supported by strong positive correlations of their mRNA expression in TCGA data. Isogenic cell clones generated by CRISPR-mediated base-editing showed that the mutations result in simultaneous upregulation of the three target genes, which were also expressed at higher levels in TCGA tumors harboring the non-coding mutations. Little is known about the function of LEPROTL1, DCTN6, and SARAF. Utilizing siRNA knockdown models complemented by live-cell imaging, viability assays, and cell cycle analysis, we found that they promote cell proliferation and cell cycle progression. Furthermore, our base-edited isogenic cell lines showed more rapid proliferation and cell cycle progression, as well as resistance to cisplatin. The mutations were also associated with shorter disease-specific survival in a TCGA cohort, and DCTN6 showed a negative association with overall survival. These results underscore the clinical significance of the non-coding mutations. Currently, we are assessing the effect of these mutations on tumor growth and cisplatin response in vivo using xenograft mouse models and evaluating the mechanisms by which LEPROTL1, DCTN6, and SARAF modulate cell cycle progression. Taken together, our data suggest that non-coding enhancer mutations near LEPROTL1 contribute to bladder carcinogenesis by increasing the expression of three co-regulated novel oncogenes, all of which were previously uncharacterized in bladder cancer. Since we found the mutations to be clinically significant, our study also raises the possibility of using the non-coding mutations and their target genes as clinical biomarkers and potential therapeutic drug targets. Citation Format: Kelly Butler, Bilal Lone, Alexandra Dobbins, Arup Chakraborty, Salah Boudajadi, Andrea Apolo, Rouf Banday. Functional and clinical consequences of hotspot non-coding mutations upstream of the LEPROTL1 gene in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A013.

Mitochondrial ACSS1-K635 acetylation knock-in mice exhibit altered metabolism, cell senescence, and nonalcoholic fatty liver disease.

Acetyl-CoA synthetase short-chain family member 1 (ACSS1) uses acetate to generate mitochondrial acetyl-CoA and is regulated by deacetylation by sirtuin 3. We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine-635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis, consistent with nonalcoholic fatty liver disease (NAFLD). RNA sequencing analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD. Fasted Acss1K635Q/K635Q mouse livers showed increased fatty acid synthase (FASN) and stearoyl-CoA desaturase 1 (SCD1), both associated with NAFLD, and increased carbohydrate response element-binding protein binding to Fasn and Scd1 enhancer regions. Last, liver lipidomics showed elevated ceramide, lysophosphatidylethanolamine, and lysophosphatidylcholine, all associated with NAFLD. Thus, we propose that ACSS1-K635-Ac dysregulation leads to aberrant lipid metabolism, cellular senescence, and NAFLD.

Abstract A028: A living biobank of patient-derived organoids reveals a role for KMT2D in maintaining luminal identity in urothelial carcinoma

Abstract Patient-derived organoid models provide a highly amenable experimental platform to study human tumor biology. Over the past seven years, our group has generated a living biobank of 82 organoid lines established from urothelial carcinomas. These organoids have been generated from all stages, are capable of unlimited expansion in vitro, and recapitulate histopathological and molecular features of their corresponding primary tumors. In addition, we have characterized the genomic, transcriptomic, and chromatin accessibility profiles of these organoid lines. Using these lines, we have identified a novel form of lineage plasticity in which a subset of organoids derived from luminal NMIBC tumors can acquire basal features in culture, but reverse back to a luminal phenotype following orthotopic xenografting in immunodeficient mice. We have focused our studies on this luminal-to-basal (L-B) plasticity since the acquisition of basal phenotypes is linked to secondary MIBC. In ongoing studies, we have examined the genomic correlates of L-B plasticity and found that loss-of-function (LOF) mutations in the epigenetic regulator KMT2D are enriched in plastic lines derived from NMIBC and in stable basal lines from basal/squamous MIBC. We also found that tumors with squamous histology in the TCGA cohort are enriched for KMT2D LOF mutations. KMT2D encodes a histone methyltransferase that adds H3K4me1 active histone marks to enhancer regions and is frequently mutated in urothelial cancers. To investigate its functional role, we performed CRISPR knock-out of KMT2D in a stable luminal organoid line and found that isogenic knock-out organoids acquired basal phenotypes. Using a barcoded lineage-tracing approach known as CellTag, we have found that acquisition of basal phenotypes occurs in a clonal fashion at the single-cell level. In parallel, we have performed a small molecule screen to identify compounds that can reverse L-B plasticity, using 17 compounds that target a range of epigenetic regulators. We found that GSK-LSD1, an inhibitor of KDM1A, can restore luminal phenotypes to plastic organoid lines in culture and increase the genomic distribution of the H3K4me1 mark, as detected by CUT&Tag. Interestingly, KDM1A is a histone demethylase that removes H3K4me1/2 marks, and thus can antagonize KMT2D. Using CellTag, we found that reversal of the basal phenotype occurs in a non-clonal fashion and represents a cell state transition at the level of single cells. In further studies, we have shown that KDM1A inhibitor treatment of fresh patient samples can prevent the L-B transition. We have also demonstrated in an organoid outgrowth assay that KDM1A inhibition can prevent invasive behavior in vitro. Moreover, an orally available KDM1A inhibitor, ORY-1001, can also reverse phenotypic plasticity, and we are currently testing its activity in xenografts to determine whether it can reverse invasive phenotypes. Overall, these results demonstrate the utility of patient-derived organoid models to investigate novel features of bladder cancer and develop therapeutic approaches. Citation Format: John R. Christin, Alana Nguyen, Talal Syed, Kwanghee Kim, Clementine Le Coz, Caroline J. Laplaca, Luis A. Pina, Reuben Akabas, Hanina Hibshoosh, Andrew T. Lenis, Guarionex J. Decastro, Christopher B. Anderson, James M. McKiernan, Chao Lu, Hikmat A. Al-Ahmadie, David B. Solit, Michael M. Shen. A living biobank of patient-derived organoids reveals a role for KMT2D in maintaining luminal identity in urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A028.

Catecholamine treatment induces reversible heart injury and cardiomyocyte gene expression

BackgroundCatecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved.ResultsC57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment.ConclusionsThe results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.

Polymorphic variants of the <i>PPP1R21</i> gene associated with the level of sex hormone-binding globulin and the risk of various stages of breast cancer

Aim. To assess the relationship of polymorphic gene variants associated with the level of sex hormone-binding globulin (SHBG) according to genome-wide association studies (GWAS) with the risk of stage I–II and stage III-IV breast cancer. Materials and methods. A comparative genetic analysis was carried out on samples of patients with breast cancer: 254 patients with stages I–II and 91 with stages III–IV, and 1140 females of the control group. The paper considers 4 single nucleotide substitutions associated with the level of circulating SHBG according to GWAS: g.107546375AG PRMT6 (rs17496332), g.27519736TC GCKR (rs780093), g.48419260TC PPP1R21 (rs10454142), g.98364050TA BAIAP2L1 (rs3779195). Results. Differences in the involvement of polymorphic variants of SHBG candidate genes in the development of stages I–II and III–IV breast cancer were revealed. The rs10454142 TC polymorphic variant in the PPP1R21 gene is associated with the risk of stage I–II breast cancer: women with an allelic variant of this locus have a higher risk of early-stage disease (T/T vs. T/C vs. C/C, odds ratio 1.35, 95% confidence interval 1.05-1.75; p=0.021; ppermutat=0.027). Also, an increase in the number of C alleles in the female genotype increased the risk of stage I–II breast cancer by 17–18% per allele. There were no associations of polymorphic variants of SHBG candidate genes with the risk of severe disease (stage III–IV). The single nucleotide substitution rs10454142 TC in the PPP1R21 gene and the single nucleotide polymorphisms strongly linked to it are functionally significant (located in the regions of enhancers and promoters) in the epithelial and myoepithelial cells of the mammary gland and liver, affect the level of genome methylation, and are associated with the level of GTF2A1L gene expression.

Yield of MR-directed US for MRI-detected breast findings: how often can we avoid MR biopsy?

PurposeTo evaluate the yield of MR-directed ultrasound for MRI detected breast findings. MethodsThis retrospective study included 857 consecutive patients who had a breast MRI between January 2017–December 2020 and received a BI-RADS 4 assessment. Only exams recommended for MR-directed ultrasound were included in the study, yielding 765 patients. Findings were characterized by presence or absence of a sonographic correlate. Utilizing the electronic medical record, for those with a sonographic correlate, the size, location, and morphology were noted. Imaging guided (Ultrasound and MRI) pathology results as well as excisional pathology results were recorded. A multivariable logistical regression analysis was used to investigate the clinical utility of MR-directed ultrasound. ResultsThere were 1262 MRI-detected BI-RADS category 4 findings in 765 patients. Of the 1262 findings, MR-directed ultrasound was performed on 852 (68 %). Of these, 291/852 (34 %) had an ultrasound correlate, including 143/291 (49 %) benign lesions, 81/291 (28 %) malignant lesions, 16/291 (5 %) with high-risk pathology and 51/291 (18 %) unknown due to lost to follow-up. Of those findings with ultrasound correlates, 173/291 (59 %) represented masses, 69/291 (24 %) were regions of non-mass enhancement, 22/291 (7.6 %) were foci and 27/291 (9.3 %) fell into the category of other which included lymph node, cysts, and scar tissue. Masses were significantly more likely to be identified on MR-directed ultrasound (p < 0.0001) compared to foci. ConclusionThe yield of MR-directed ultrasound is significantly higher for masses, than foci and non-mass enhancement, which should be taken into consideration when recommending an MR-directed ultrasound.

Exploring the epigenetic profile of ID4 in breast cancer: bioinformatic insights into methylation patterns and chromatin accessibility dynamics.

Inhibitor of differentiation 4 (ID4) is a dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors. The expression of ID4 is dysregulated in various breast cancer subtypes, indicating a potential role for ID4 in subtype-specific breast cancer development. This study aims to elucidate the epigenetic regulation of ID4 within breast cancer subtypes, with a particular focus on DNA methylation and chromatin accessibility. Bioinformatic analyses were conducted to assess DNA methylation and chromatin accessibility in ID4 regulatory regions across breast cancer subtypes. Gene Set Enrichment Analysis (GSEA) was conducted to identify related gene sets. Transcription factor binding within ID4 enhancer and promoter regions was explored. In vitro experiments involved ER+ breast cancer cell lines treated with estradiol (E2) and Tamoxifen. Distinct epigenetic profiles of ID4 were observed, revealing increased methylation and reduced chromatin accessibility in luminal subtypes compared to the basal subtype. Gene Set Enrichment Analysis (GSEA) implicated estrogen-related pathways, suggesting a potential link between estrogen signaling and the regulation of ID4 expression. Transcription factor analysis identified ER and FOXA1 as regulators of ID4 enhancer regions. In vitro experiments confirmed the role of ER, demonstrating reduced ID4 expression and increased methylation with estradiol treatment. Conversely, Tamoxifen treatment increased ID4 expression, indicating the potential involvement of ER signaling through ERα in the epigenetic regulation of ID4 in breast cancer cells. This study shows the intricate epigenetic regulation of ID4 in breast cancer, highlighting subtype-specific differences in DNA methylation and chromatin accessibility.

Drosophila TET acts with PRC1 to activate gene expression independently of its catalytic activity.

Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Hold the MYCrophone: MYC Invades Enhancers to Control Cancer-Type Gene Programs.

MYC is an oncogenic transcription factor that binds gene promoters to facilitate oncogenic gene expression. When overexpressed, as is the case in most human cancers, MYC also invades active enhancers-cis-regulatory elements that are critical for regulating gene expression. In previous studies, the regulatory significance of MYC enhancer invasion in cancer cells has been debated. In their study published in Nature Genetics, Jakobsen and colleagues establish a new role for MYC in enhancer regions: regulating cancer type-specific gene programs. Their work reveals a mechanism in which MYC cooperates with other oncogenic transcription factors to recruit epigenetic regulators to enhancers, resulting in an epigenetic "switch" that promotes enhancer activation through BRD4 and RNA polymerase II. This activity was highly cancer-type specific, highlighting gene expression programs that predicted clinical outcome in a subtype-specific manner in patients with breast cancer.

Novel putative causal mutations associated with fat traits in Nellore cattle uncovered by eQTLs located in open chromatin regions

Intramuscular fat (IMF) and backfat thickness (BFT) are critical economic traits impacting meat quality. However, the genetic variants controlling these traits need to be better understood. To advance knowledge in this area, we integrated RNA-seq and single nucleotide polymorphisms (SNPs) identified in genomic and transcriptomic data to generate a linkage disequilibrium filtered panel of 553,581 variants. Expression quantitative trait loci (eQTL) analysis revealed 36,916 cis-eQTLs and 14,408 trans-eQTLs. Association analysis resulted in three eQTLs associated with BFT and 24 with IMF. Functional enrichment analysis of genes regulated by these 27 eQTLs revealed noteworthy pathways that can play a fundamental role in lipid metabolism and fat deposition, such as immune response, cytoskeleton remodeling, iron transport, and phospholipid metabolism. We next used ATAC-Seq assay to identify and overlap eQTL and open chromatin regions. Six eQTLs were in regulatory regions, four in predicted insulators and possible CCCTC-binding factor DNA binding sites, one in an active enhancer region, and the last in a low signal region. Our results provided novel insights into the transcriptional regulation of IMF and BFT, unraveling putative regulatory variants.

Evolutionary Rate Shifts in Coding and Regulatory Regions Underpin Repeated Adaptation to Sulfidic Streams in Poeciliid Fishes.

Adaptation to extreme environments often involves the evolution of dramatic physiological changes. To better understand how organisms evolve these complex phenotypic changes, the repeatability and predictability of evolution, and possible constraints on adapting to an extreme environment, it is important to understand how adaptive variation has evolved. Poeciliid fishes represent a particularly fruitful study system for investigations of adaptation to extreme environments due to their repeated colonization of toxic hydrogen sulfide-rich springs across multiple species within the clade. Previous investigations have highlighted changes in the physiology and gene expression in specific species that are thought to facilitate adaptation to hydrogen sulfide-rich springs. However, the presence of adaptive nucleotide variation in coding and regulatory regions and the degree to which convergent evolution has shaped the genomic regions underpinning sulfide tolerance across taxa are unknown. By sampling across seven independent lineages in which nonsulfidic lineages have colonized and adapted to sulfide springs, we reveal signatures of shared evolutionary rate shifts across the genome. We found evidence of genes, promoters, and putative enhancer regions associated with both increased and decreased convergent evolutionary rate shifts in hydrogen sulfide-adapted lineages. Our analysis highlights convergent evolutionary rate shifts in sulfidic lineages associated with the modulation of endogenous hydrogen sulfide production and hydrogen sulfide detoxification. We also found that regions with shifted evolutionary rates in sulfide spring fishes more often exhibited convergent shifts in either the coding region or the regulatory sequence of a given gene, rather than both.

LINC00887 Acts as an Enhancer RNA to Promote Medullary Thyroid Carcinoma Progression by Binding with FOXQ1.

Medullary thyroid carcinoma (MTC) is a rare but aggressive endocrine malignancy that originates from the parafollicular C cells of the thyroid gland. Enhancer RNAs (eRNAs) are non-coding RNAs transcribed from enhancer regions, which are critical regulators of tumorigenesis. However, the roles and regulatory mechanisms of eRNAs in MTC remain poorly understood. This study aims to identify key eRNAs regulating the malignant phenotype of MTC and to uncover transcription factors involved in the regulation of key eRNAs. GSE32662 and GSE114068 were used for the identification of differentially expressed genes, eRNAs, enhancers and enhancer-regulated genes in MTC. Metascape and the transcription factor affinity prediction method were used for gene function enrichment and transcription factor prediction, respectively. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to assess the binding of histone H3 lysine 27 acetylation (H3K27ac)-enriched regions to anti- H3K27ac. RIP-qPCR was used to detect the binding between FOXQ1 and LINC00887. CCK8 and Transwell were performed to measure the proliferation and invasion of MTC cells, respectively. Intracellular reactive oxygen species (ROS) levels were quantified using a ROS assay kit. Four eRNAs (H1FX-AS1, LINC00887, MCM3AP-AS1 and A1BG-AS1) were screened, among which LINC00887 was the key eRNA promoting the proliferation and invasion of MTC cells. A total of 135 genes controlled by LINC00887-regulated enhancers were identified; among them, BCL2, PRDX1, SFTPD, TPO, GSS, RAD52, ZNF580, and ZFP36L1 were significantly enriched in the "ROS metabolic process" term. As a transcription factor regulating genes enriched in the "ROS metabolic process" term, FOXQ1 could recruit LINC00887. Overexpression of FOXQ1 restored LINC00887 knockdown-induced downregulation of GSS and ZFP36L1 transcription in MTC cells. Additionally, FOXQ1 overexpression counteracted the inhibitory effects of LINC00887 knockdown on the proliferation and invasion of MTC cells and the promotion of intracellular ROS accumulation induced by LINC00887 knockdown. LINC00887 was identified as a key eRNA promoting the malignant phenotype of MTC cells. The involvement of FOXQ1 was essential for LINC00887 to play a pro-tumorigenic role in MTC. Our findings suggest that the FOXQ1/LINC00887 axis is a potential therapeutic target for MTC.

Abstract 150: Decoding The Variant-to-function Relationship For LIPA , A Risk Locus For Cad

Background: Genome-wide association studies (GWASs) link genetic variation in the LIPA (lysosomal acid lipase) gene to coronary artery disease (CAD). Quantitative Trait Loci (QTL) studies indicate higher LIPA mRNA and enzyme activity in the monocytes of risk alleles carriers. Yet, the mechanism whereby this locus confers disease etiology has not been fully established. Herein, we aim to establish the variant-to-function relationship and elucidate how increased LIPA impacts atherosclerosis. Methods: Employing post-GWAS pipelines (eQTL, enzyme activity-QTL, Tri-HiC, luciferase assay, allele-specific binding, motif analysis, and mobility shift assay), we connect functional variants to target genes in causal myeloid cells. Myeloid-specific Lipa overexpression mice ( Lipa Tg ) on Ldlr -/- background were generated and characterized. Results: Post-GWAS pipelines support LIPA as the candidate causal gene at this locus. In human monocyte-derived macrophages, risk allele carriers of GWAS CAD variants exhibit higher LIPA mRNA, protein, and enzyme activity. Tri-HiC and luciferase assay identified an intronic region within LIPA , housing GWAS CAD noncoding variants and exhibiting monocyte-specific enhancer activity. Within the enhancer region, 1412445 and rs1320496 were identified as the two potential causal variants, with the risk alleles having increased enhancer activity in luciferase assay and increased binding to PU.1 in mobility shift assay. Collectively, the above data suggest that the risk alleles of CAD variants lead to increased LIPA in monocytes/macrophages. Consistently, Lipa Tg , Ldlr -/- mice fed a Western diet showed significantly increased lesion size and lesion macrophage area compared to Ctl, Ldlr -/- mice. scRNA-seq analysis followed by functional validation demonstrated that Lipa Tg macrophages had higher free cholesterol yet lower neutral lipid accumulation, accompanied by a transcriptomic signature of increased interferon signaling. Lipa overexpression also increased monocyte infiltration into the plaque. Conclusions: We established that the risk alleles of common noncoding variants at the LIPA locus drive increased myeloid LIPA expression and aggravate atherosclerosis.

Abstract 1099: Single-cell Transcriptomic And Epigenic Profiling Identifies Sox6 As A Key Regulator Of Arterial Endothelial Cell Phenotype In Systemic Sclerosis-associated Pulmonary Hypertension

Background: Pulmonary hypertension (PH) frequently arises as a complication of systemic sclerosis (SSc), and is a major cause of death among patients with this condition. Pulmonary vasculopathy in SSc is characterized by endothelial dysfunction with subsequent remodeling of the pulmonary vasculature, especially arterioles and small arteries. Characterization of transcriptional regulators driving the pathological changes in arterial endothelial cells (ECs) is critical to understanding pathogenic mechanisms underlying SSc-associated vasculopathy. Methods and Results: We performed single-cell multiome sequencing to simultaneously profile the transcriptome and epigenome of explanted lung tissue from SSc-PH patients (n = 9) and healthy donors (n = 6). We identified co-regulated genes and candidate enhancer regions associated with transcription factors (TFs) across different EC cell states using SCENIC+. Gene regulatory network analysis revealed SOX6 as a key transcription factor for the arterial EC phenotype associated with SSc-PH. 71 genes and 153 regions were predicted to be regulated by SOX6. Notably, SOX6 was the only SOX family member significantly upregulated in SSc-PH arterial ECs (fold change = 40.0, adjusted p-value = 6.1*10 -21 ). Putative bound TF motif instances accounting for chromatin accessibility in the candidate enhancer regions were identified using the base-resolution deep learning model, ChromBPNet. Consistent with SCENIC+ predictions, cell-state specific ChromBPNet models revealed higher putative TF occupancy at SOX motif instances across the genome. In addition, we identified SOX/ETS composite motif instances showing higher putative occupancy in SSc-PH arterial ECs, which may support cooperative binding of SOX proteins with ETS family members. Conclusions: These data provide novel insight into transcriptional regulatory programs of arterial ECs in SSc-PH and highlight SOX6 as a key contributor.

Abstract 1014: Molecular Dynamics In The Progression Of Aortic Dissection With Emphasis On De Novo Elastic Fiber Synthesis

Background: Aortic dissection (AD) confined to the descending thoracic aorta is typically managed conservatively in the acute phase. However, these patients often require surgeries due to aortic dilatation in the chronic phase. Although preclinical studies have been conducted to investigate the mechanism of AD, most studies focused on the acute phase, especially the cause of AD. It remains unknown how molecular mechanisms alter following AD. Methods and Results: To determine the molecular alteration after AD, bulk RNA sequencing was performed using aortas from mice administered β-aminopropionitrile (BAPN) for 1, 4, or 12 weeks that represent pre, acute, and chronic phases of ADs, respectively. There were 6,755 differentially expressed genes (DEGs), and 2,667 and 1,173 DEGs were upregulated in the acute and chronic phases, respectively. Gene ontology analysis revealed that DEGs in the acute phase were related to inflammation, whereas DEGs in the chronic phase were associated with extracellular matrix. Those DEGs in the chronic phase included tropoelastin ( Eln ), the precursor of elastic fibers. In situ hybridization and immunostaining demonstrated that Eln mRNA and protein were distributed in the vascular wall of the false lumen. Of note, these molecules formed new elastic fibers, as evidenced by Verhoeff’s iron hematoxylin staining. The de novo fibers were thinner than native fibers; however, ultrasonography revealed that aortas with AD lesions had higher elasticity and distensibility compared to intact aortas. These data suggest that new elastic fibers protect against aortic rupture after AD. Next, a transcriptional factor prediction analysis was performed to identify a potential regulator of Eln transcription and found that Etv5 could be aligned in both the promoter and enhancer regions of Eln . Immunostaining showed that ETV5 was spatially coincident with Eln mRNA and de novo fibers. Conclusion: Genes related to the extracellular matrix are upregulated in response to AD in BAPN-administered mice. Elastic fibers are newly formed in the vascular wall of the false lumen in chronic AD accompanied by enhanced Etv5 .

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD)

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.

Germline cis variant determines epigenetic regulation of the anti-cancer drug metabolism gene dihydropyrimidine dehydrogenase (DPYD).

Enhancers are critical for regulating tissue-specific gene expression, and genetic variants within enhancer regions have been suggested to contribute to various cancer-related processes, including therapeutic resistance. However, the precise mechanisms remain elusive. Using a well-defined drug-gene pair, we identified an enhancer region for dihydropyrimidine dehydrogenase (DPD, DPYD gene) expression that is relevant to the metabolism of the anti-cancer drug 5-fluorouracil (5-FU). Using reporter systems, CRISPR genome-edited cell models, and human liver specimens, we demonstrated in vitro and vivo that genotype status for the common germline variant (rs4294451; 27% global minor allele frequency) located within this novel enhancer controls DPYD transcription and alters resistance to 5-FU. The variant genotype increases recruitment of the transcription factor CEBPB to the enhancer and alters the level of direct interactions between the enhancer and DPYD promoter. Our data provide insight into the regulatory mechanisms controlling sensitivity and resistance to 5-FU.