Enriched Transcription Factor Research Articles

Innovative breakthroughs facilitated by single-cell multi-omics: manipulating natural killer cell functionality correlates with a novel subcategory of melanoma cells.

Melanoma is typically regarded as the most dangerous form of skin cancer. Although surgical removal of in situ lesions can be used to effectively treat metastatic disease, this condition is still difficult to cure. Melanoma cells are removed in great part due to the action of natural killer (NK) and T cells on the immune system. Still, not much is known about how the activity of NK cell-related pathways changes in melanoma tissue. Thus, we performed a single-cell multi-omics analysis on human melanoma cells in this study to explore the modulation of NK cell activity. Cells in which mitochondrial genes comprised > 20% of the total number of expressed genes were removed. Gene ontology (GO), gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and AUCcell analysis of differentially expressed genes (DEGs) in melanoma subtypes were performed. The CellChat package was used to predict cell-cell contact between NK cell and melanoma cell subtypes. Monocle program analyzed the pseudotime trajectories of melanoma cells. In addition, CytoTRACE was used to determine the recommended time order of melanoma cells. InferCNV was utilized to calculate the CNV level of melanoma cell subtypes. Python package pySCENIC was used to assess the enrichment of transcription factors and the activity of regulons in melanoma cell subtypes. Furthermore, the cell function experiment was used to confirm the function of TBX21 in both A375 and WM-115 melanoma cell lines. Following batch effect correction, 26,161 cells were separated into 28 clusters and designated as melanoma cells, neural cells, fibroblasts, endothelial cells, NK cells, CD4+ T cells, CD8+ T cells, B cells, plasma cells, monocytes and macrophages, and dendritic cells. A total of 10137 melanoma cells were further grouped into seven subtypes, i.e., C0 Melanoma BIRC7, C1 Melanoma CDH19, C2 Melanoma EDNRB, C3 Melanoma BIRC5, C4 Melanoma CORO1A, C5 Melanoma MAGEA4, and C6 Melanoma GJB2. The results of AUCell, GSEA, and GSVA suggested that C4 Melanoma CORO1A may be more sensitive to NK and T cells through positive regulation of NK and T cell-mediated immunity, while other subtypes of melanoma may be more resistant to NK cells. This suggests that the intratumor heterogeneity (ITH) of melanoma-induced activity and the difference in NK cell-mediated cytotoxicity may have caused NK cell defects. Transcription factor enrichment analysis indicated that TBX21 was the most important TF in C4 Melanoma CORO1A and was also associated with M1 modules. In vitro experiments further showed that TBX21 knockdown dramatically decreases melanoma cells' proliferation, invasion, and migration. The differences in NK and T cell-mediated immunity and cytotoxicity between C4 Melanoma CORO1A and other melanoma cell subtypes may offer a new perspective on the ITH of melanoma-induced metastatic activity. In addition, the protective factors of skin melanoma, STAT1, IRF1, and FLI1, may modulate melanoma cell responses to NK or T cells.

A Rare Olive Compound Oleacein Improves Lipid and Glucose Metabolism, and Inflammatory Functions: A Comprehensive Whole-Genome Transcriptomics Analysis in Adipocytes Differentiated from Healthy and Diabetic Adipose Stem Cells.

Oleacein (OLE), a rare natural compound found in unfiltered extra virgin olive oil, has been shown to have anti-inflammatory and anti-obesity properties. However, little is known regarding the mechanisms by which OLE influences metabolic processes linked to disease targets, particularly in the context of lipid metabolism. In the present study, we conducted whole-genome DNA microarray analyses in adipocytes differentiated from human adipose-derived stem cells (hASCs) and diabetic hASCs (d-hASCs) to examine the effects of OLE on modulating metabolic pathways. We found that OLE significantly inhibited lipid formation in adipocytes differentiated from both sources. In addition, microarray analysis demonstrated that OLE treatment could significantly downregulate lipid-metabolism-related genes and modulate glucose metabolism in both adipocyte groups. Transcription factor enrichment and protein-protein interaction (PPI) analyses identified potential regulatory gene targets. We also found that OLE treatment enhanced the anti-inflammatory properties in adipocytes. Our study findings suggest that OLE exhibits potential benefits in improving lipid and glucose metabolism, thus holding promise for its application in the management of metabolic disorders.

1737-P: Novel Interaction between Nkx6.1 and Pdx1 Is Needed for Proper Beta-Cell Function

The beta cell transcription factor Nkx6.1 is critical in pancreatic progenitor cells and mature beta cells. The pathways regulated by Nkx6.1 that results in these phenotypes are still being defined. We sought to define the transcriptional factors that interact with Nkx6.1 and may be harnessed to expand functional beta cell mass. INS-1 832/13 beta cells were transduced with AdCMV-Nkx6.1-BioID, AdCMV-GFP-BioID or left untreated. Biotinylated proteins were isolated and the Nkx6.1 interactome was defined using liquid chromatography-tandem mass spectrometry. We defined 176 biotinylated proteins in cells transduced with AdCMV-Nkx6.1-BioID that were not observed with AdCMV-GFP-BioID or naturally biotinylated proteins from untreated lysates. These potential Nkx6.1 interacting proteins are enriched in nucleotide binding, ribosomal proteins, and transcription factors. Among the enriched transcription factors, we validated that Pdx1 is biotinylated in response to transduction with AdCMV-Nkx6.1 using biotin-targeted immunoprecipitation and western blot. To examine if Pdx1 is likely to interact with Nkx6.1 on the genome, we analyzed the frequency of proximal binding sites for Nkx6.1 and Pdx1, and demonstrated that Pdx1 and Nkx6.1 binding sites in close proximity were statistically overrepresented in the genome. Given the role of Pdx1 on functional beta cell mass, we further characterized the interaction of endogenous Nkx6.1 and Pdx1 by co-immunoprecipitation (Co-IP). Our data demonstrates significant Pdx1 binding by Nkx6.1 Co-IP and significant Nkx6.1 binding by Pdx1 Co-IP, as compared to Co-IP with the IgG control. Finally, we demonstrate coregulation of Unc3 expression by Nkx6.1 and Pdx1 and demonstrate that CRISPR mediated deletion of either Nkx6.1 or Pdx1 is sufficient to impair INS-1 832/13 mediated expression of Unc3. These data define potential Nkx6.1 binding partners in INS-1 832/13 cells, as well as novel interaction between Nkx6.1 and Pdx1 in the regulation of Unc3. Disclosure J.S.Tessem: None. C.C.Littlefield: None. J.T.Hill: None.

1779-P: Lipotoxicity-Induced Beta-Cell Small Extracellular Vesicles Promote Beta-Cell Functional Failure through Activation of the TGFß/Smad3 Pathway

Lipotoxicity is a pathological process that occurs in several metabolic disorders including obesity-driven Type 2 diabetes (T2D). Chronically elevated circulating free fatty acids have been shown to exert detrimental effects on pancreatic islet β-cells through reduced glucose-stimulated insulin secretion (GSIS), alterations in β-cell transcriptional identity, and apoptosis. β-cell-derived small extracellular vesicles (sEV) have been shown to contribute to β-cell failure in T2D, however their role in lipotoxicity-mediated β-cell failure has yet to be elucidated. We hypothesize that lipotoxicity-induced β-cell functional failure is mediated in part by aberrant release of altered β-cell-derived sEV. To test this, MIN6 β-cells were exposed to free fatty acid, palmitate (PAL) (0.5 mM, 24h) and EV were isolated using differential ultracentrifugation to yield PAL EV (vs. control (CTL) EV). β-cell functional assessment using GSIS on mouse islets exposed to PAL EV (48h) resulted in significant suppression of GSIS (~80% decrease in stimulation index). RNA-Seq was conducted on islets exposed to PAL EV (vs. untreated (UT)). De novo motif analysis from this data identified enriched transcription factor binding motifs associated with several SMAD genes in TGFβ signaling. Gene Set Enrichment Analysis revealed significant enrichment of genes associated with TGFβ receptor binding suggesting its potential role in lipotoxic β-cell EV-mediated β-cell failure. To confirm, analysis of p-Smad3 expression was conducted by immunofluorescence staining on mouse islets exposed to PAL EV (48h; vs. UT). The results showed an ~60-fold increase in β-cell p-Smad3 expression (p<.05). Moreover, mouse islets were treated with a TGFβ receptor inhibitor in the presence of PAL EV (48h; vs. UT). GSIS results showed a significant improvement in β-cell function (p<.05) suggesting the role of the TGFβ/Smad3 pathway in lipotoxic β-cell sEV induced β-cell failure. Disclosure A.Roy: None. A.Hoff: None. T.K.Her: None. K.Sankaran rajagopalan: None. D.D.Maltais: None. A.Matveyenko: None. N.Javeed: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK129208-01); JDRF (2-SRA-2022-1272-S-B)

Determination of steady-state transcriptome modifications associated with repeated homotypic stress in the rat rostral posterior hypothalamic region.

Chronic stress is epidemiologically correlated with physical and psychiatric disorders. Whereas many animal models of chronic stress induce symptoms of psychopathology, repeated homotypic stressors to moderate intensity stimuli typically reduce stress-related responses with fewer, if any, pathological symptoms. Recent results indicate that the rostral posterior hypothalamic (rPH) region is a significant component of the brain circuitry underlying response reductions (habituation) associated with repeated homotypic stress. To test whether posterior hypothalamic transcriptional regulation associates with the neuroendocrine modifications induced by repeated homotypic stress, RNA-seq was performed in the rPH dissected from adult male rats that experienced either no stress, 1, 3, or 7 stressful loud noise exposures. Plasma samples displayed reliable increases of corticosterone in all stressed groups, with the smallest increase in the group exposed to 7 loud noises, indicating significant habituation compared to the other stressed groups. While few or no differentially expressed genes were detected 24-h after one or three loud noise exposures, relatively large numbers of transcripts were differentially expressed between the group exposed to 7 loud noises when compared to the control or 3-stress groups, respectively, which correlated with the corticosterone response habituation observed. Gene ontology analyses indicated multiple significant functional terms related to neuron differentiation, neural membrane potential, pre- and post-synaptic elements, chemical synaptic transmission, vesicles, axon guidance and projection, glutamatergic and GABAergic neurotransmission. Some of the differentially expressed genes (Myt1l, Zmat4, Dlx6, Csrnp3) encode transcription factors that were independently predicted by transcription factor enrichment analysis to target other differentially regulated genes in this study. A similar experiment employing in situ hybridization histochemical analysis in additional animals validated the direction of change of the 5 transcripts investigated (Camk4, Gabrb2, Gad1, Grin2a and Slc32a) with a high level of temporal and regional specificity for the rPH. In aggregate, the results suggest that distinct patterns of gene regulation are obtained in response to a repeated homotypic stress regimen; they also point to a significant reorganization of the rPH region that may critically contribute to the phenotypic modifications associated with repeated homotypic stress habituation.

Uncovering neuroinflammation-related modules and potential repurposing drugs for Alzheimer's disease through multi-omics data integrative analysis.

Neuroinflammation is one of the key factors leading to neuron death and synapse dysfunction in Alzheimer's disease (AD). Amyloid-β (Aβ) is thought to have an association with microglia activation and trigger neuroinflammation in AD. However, inflammation response in brain disorders is heterogenous, and thus, it is necessary to unveil the specific gene module of neuroinflammation caused by Aβ in AD, which might provide novel biomarkers for AD diagnosis and help understand the mechanism of the disease. Transcriptomic datasets of brain region tissues from AD patients and the corresponding normal tissues were first used to identify gene modules through the weighted gene co-expression network analysis (WGCNA) method. Then, key modules highly associated with Aβ accumulation and neuroinflammatory response were pinpointed by combining module expression score and functional information. Meanwhile, the relationship of the Aβ-associated module to the neuron and microglia was explored based on snRNA-seq data. Afterward, transcription factor (TF) enrichment and the SCENIC analysis were performed on the Aβ-associated module to discover the related upstream regulators, and then a PPI network proximity method was employed to repurpose the potential approved drugs for AD. A total of 16 co-expression modules were primarily obtained by the WGCNA method. Among them, the green module was significantly correlated with Aβ accumulation, and its function was mainly involved in neuroinflammation response and neuron death. Thus, the module was termed the amyloid-β induced neuroinflammation module (AIM). Moreover, the module was negatively correlated with neuron percentage and showed a close association with inflammatory microglia. Finally, based on the module, several important TFs were recognized as potential diagnostic biomarkers for AD, and then 20 possible drugs including ibrutinib and ponatinib were picked out for the disease. In this study, a specific gene module, termed AIM, was identified as a key sub-network of Aβ accumulation and neuroinflammation in AD. Moreover, the module was verified as having an association with neuron degeneration and inflammatory microglia transformation. Moreover, some promising TFs and potential repurposing drugs were presented for AD based on the module. The findings of the study shed new light on the mechanistic investigation of AD and might make benefits the treatment of the disease.

Unearths IFNB1 immune infiltrates in SOP-related ossification of ligamentum flavum pathogenesis

BackgroundOssification of ligamentum flavum (OLF) is a hidden, indolent disease condition with variable unexplained etiology and pathology. Growing evidences show a correlation between senile osteoporosis (SOP) and OLF, but the fundamental relationship between SOP and OLF remains unclear. Therefore, the purpose of this work is to investigate unique SOP-related genes and their potential functions in OLF. MethodsGene Expression Omnibus (GEO) database was utilized to gather the mRNA expression data (GSE106253) and then analyzed by R software. A variety of methods, including ssGSEA, machine learning (LASSO and SVM-RFE), GO and KEGG enrichment, PPI network, transcription factor enrichment analysis (TFEA), GSEA and xCells were employed to verified the critical genes and signaling pathways. Furthermore, ligamentum flavum cells were cultured and used in vitro to identify the expression of the core genes. ResultsThe preliminary identification of 236 SODEGs revealed their involvement in BP pathways associated with ossification, inflammation, and immune response, including the TNF signaling pathway, PI3K/AKT signaling pathway and osteoclast differentiation. Four down-regulated genes (SERPINE1, SOCS3, AKT1, CCL2) and one up-regulated gene (IFNB1) were among the five hub SODEGs that were validated. Additionally, they were performed by ssGSEA and xCell to show the relationship of immune cells infiltrating in OLF. The most fundamental gene, IFNB1, which was only found in the classical ossification- and inflammation-related pathways, suggested that it may affect OLF via regulating the inflammatory response. In vitro experiment, we found that IFNB1 expression was dramatically higher in cells cocultured with osteogenic induction than in controls. ConclusionAs far as we are concerned, this is the first observation using transcriptome data mining to reveal distinct SOP-related gene profiles between OLF and normal controls. Five hub SODEGs were ultimately found using bioinformatics algorithms and experimental verification. These genes may mediate intricate inflammatory/immune responses or signaling pathways in the pathogenesis of OLF, according to the thorough functional annotations. Since IFNB1 was discovered to be a key gene and was connected to numerous immune infiltrates in OLF, it is possible that IFNB1 expression has a substantial impact on the pathogenesis of OLF. Our research will give rise to new possibilities for potential therapeutics that target SOP reverent genes and immune-associated pathways in OLF.

CenhANCER: a comprehensive cancer enhancer database for primary tissues and cell lines.

Enhancers, which are key tumorigenic factors with wide applications for subtyping, diagnosis and treatment of cancer, are attracting increasing attention in the cancer research. However, systematic analysis of cancer enhancers poses a challenge due to the lack of integrative data resources, especially those from tumor primary tissues. To provide a comprehensive enhancer profile across cancer types, we developed a cancer enhancer database CenhANCER by curating public resources including all the public H3K27ac ChIP-Seq data from 805 primary tissue samples and 671 cell line samples across 41 cancer types. In total, 57 029 408 typical enhancers, 978 411 super-enhancers and 226 726 enriched transcription factors were identified. We annotated the super-enhancers with chromatin accessibility regions, cancer expression quantitative trait loci (eQTLs), genotype-tissue expression eQTLs and genome-wide association study risk single nucleotide polymorphisms (SNPs) for further functional analysis. The identified enhancers were highly consistent with accessible chromatin regions in the corresponding cancer types, and all the 10 super-enhancer regions identified from one colorectal cancer study were recapitulated in our CenhANCER, both of which testified the high quality of our data. CenhANCER with high-quality cancer enhancer candidates and transcription factors that are potential therapeutic targets across multiple cancer types provides a credible resource for single cancer analysis and for comparative studies of various cancer types. Database URL http://cenhancer.chenzxlab.cn/.

Network-based analysis identifies key regulatory transcription factors involved in skin aging

Skin aging is a complex process involving intricate genetic and environmental factors. In this study, we performed a comprehensive analysis of the transcriptional regulatory landscape of skin aging in canines. Weighted Gene Co-expression Network Analysis (WGCNA) was employed to identify aging-related gene modules. We subsequently validated the expression changes of these module genes in single-cell RNA sequencing (scRNA-seq) data of human aging skin. Notably, basal cell (BC), spinous cell (SC), mitotic cell (MC), and fibroblast (FB) were identified as the cell types with the most significant gene expression changes during aging. By integrating GENIE3 and RcisTarget, we constructed gene regulation networks (GRNs) for aging-related modules and identified core transcription factors (TFs) by intersecting significantly enriched TFs within the GRNs with hub TFs from WGCNA analysis, revealing key regulators of skin aging. Furthermore, we demonstrated the conserved role of CTCF and RAD21 in skin aging using an H2O2-stimulated cell aging model in HaCaT cells. Our findings provide new insights into the transcriptional regulatory landscape of skin aging and unveil potential targets for future intervention strategies against age-related skin disorders in both canines and humans.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Substantiate a read-across hypothesis by using transcriptome data-A case study on volatile diketones.

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, β-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24h and 72h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and β-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to β-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.

Identification of long noncoding RNAs with aberrant expression in prostate cancer metastases.

Prostate cancer (PCa) is the second-most common cause of male cancer-related death in western industrialized countries, and the emergence of metastases is a key challenge in the treatment of PCa. Accumulating studies have shown that long noncoding RNAs (lncRNAs) play an important role in the regulation of diverse cellular and molecular processes during the development and progression of cancer. Here, we utilized a unique cohort of castration-resistant prostate cancer metastases (mCRPC) and corresponding localized tumors and RNA sequencing (RNA-seq). First, we showed that patient-to-patient variability accounted for most of the variance in lncRNA expression between the samples, suggesting that genomic alterations in the samples are the main drivers of lncRNA expression in PCa metastasis. Subsequently, we identified 27 lncRNAs with differential expression (DE-lncRNAs) between metastases and corresponding primary tumors, suggesting that they are mCRPC-specific lncRNAs. Analyses of potential regulation by transcription factors (TFs) revealed that approximately half of the DE-lncRNAs have at least one binding site for the androgen receptor in their regulatory regions. In addition, TF enrichment analysis revealed the enrichment of binding sites for PCa-associated TFs, such as FOXA1 and HOXB13, in the regulatory regions of the DE-lncRNAs. In a cohort of prostatectomy-treated prostate tumors, four of the DE-lncRNAs showed association with progression-free time and two of them (lnc-SCFD2-2 and lnc-R3HCC1L-8) were independent prognostic markers. Our study highlights several mCRPC-specific lncRNAs that might be important in the progression of the disease to the metastatic stage and may also serve as potential biomarkers for aggressive PCa.

A Descriptive Whole-Genome Transcriptomics Study in a Stem Cell-Based Tool Predicts Multiple Tissue-Specific Beneficial Potential and Molecular Targets of Carnosic Acid.

Carnosic acid (CA) is a phenolic diterpene widely distributed in herbal plants, rosemary and sage. Although its medicinal properties, such as antioxidant, antimicrobial, and neuroprotective effects, have been well-documented, its relevant biochemical processes and molecular targets have not been fully explored yet. In the present study, we conducted an untargeted whole-genome transcriptomics analysis to investigate CA-induced early biological and molecular events in human amniotic epithelial stem cells (hAESCs) with the aim of exploring its multiple tissue-specific functionalities and potential molecular targets. We found that seven days of CA treatment in hAESCs could induce mesoderm-lineage-specific differentiation. Tissue enrichment analysis revealed that CA significantly enriched lateral plate mesoderm-originated cardiovascular and adipose tissues. Further tissue-specific PPI analysis and kinase and transcription factor enrichment analyses identified potential upstream regulators and molecular targets of CA in a tissue-specific manner. Gene ontology enrichment analyses revealed the metabolic, antioxidant, and antifibrotic activities of CA. Altogether, our comprehensive whole-genome transcriptomics analyses offer a thorough understanding of the possible underlying molecular mechanism of CA.

Analysis of basic pentacysteine6 transcription factor involved in abiotic stress response in Arabidopsis thaliana.

Background: Abiotic stress is a significant environmental factor that limits plant growth. Plants have complex and diverse mechanisms for dealing with abiotic stress, and different response mechanisms are interconnected. Our research aims to find key transcription factors that can respond to multiple non -biological stress. Methods: We used gene expression profile data of Arabidopsis in response to abiotic stress, constructed a weighted gene co-expression network, to obtain key modules in the network. The functions and pathways involved in these modules were further explored by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Through the enrichment analysis of transcription factor, the transcription factor that plays an important regulatory role in the key module. Through gene difference expression analysis and building protein interaction networks, the important role of key transcription factors is verified. Result: In weighted gene co-expression network, identified three gene modules that are primarily associated with cold stress, heat stress, and salt stress. Functional enrichment analysis indicated that the genes in these modules participate in biological processes such as protein binding, stress response, and others. Transcription factor enrichment analysis revealed that the transcription factor Basic Pentacysteine6 (BPC6) plays a crucial regulatory role in these three modules. The expression of the BPC6 gene is dramatically affected under a variety of abiotic stress treatments, according to an analysis of Arabidopsis gene expression data under abiotic stress treatments. Differential expression analysis showed that there were 57 differentially expressed genes in bpc4 bpc6 double mutant Arabidopsis relative to normal Arabidopsis samples, including 14 BPC6 target genes. Protein interaction network analysis indicated that the differentially expressed genes had strong interactions with BPC6 target genes within the key modules. Conclusion: Our findings reveal that the BPC6 transcription factor plays a key regulatory function in Arabidopsis coping with a variety of abiotic stresses, which opens up new ideas and perspectives for us to understand the mechanism of plants coping with abiotic stresses.

Abstract 1484: Identification of the critical active enhancer and regulated transcripts contributing to the refractory nature of pancreatic cancer

Abstract Background: Pancreatic adenocarcinoma (PAAD) is the most lethal human cancer, with a 5-year survival rate of less than 10%. Thus, the identification of prognostic biomarkers as well as molecular therapeutic targets should be critical. Gene expression is regulated by various factors such as transcription factors, promoters, enhancers, DNA methylation, microRNAs, etc. Among them, it has been reported that enhancers play an essential role in the development and progression of cancer. This study aims to identify the novel regulatory element of gene transcription and therapeutic targets in pancreatic cancer by analyzing enhancers. Material and Methods: We implemented NET-CAGE analysis to identify the active enhancer regions in pancreatic cancer cell lines and two immortalized normal pancreatic cell lines. As for the enhancer sequences, we conducted a motif analysis using an analytical pipeline, HOMER to capture sequence motifs enriched in the enhancers. The p-values output by HOMER were applied to identify transcription factor motifs enriched in the tumor cell lines compared to the normal cell lines. We analyzed RNA-sequencing data derived not only from 105 cases of in-house pancreatic cancer but from 178 cases of The Cancer Genome Atlas. Results: We found that the enrichment of KLF and ELF family transcription factors was observed in cancer cell lines compared to normal cell lines. ELF4 expression was significantly higher in tumor tissues than in normal tissues (Wilcoxon rank sum test, p<0.05). In survival analysis, high ELF4 expression was significantly associated with poor prognosis (Kaplan-Meier method, Log-rank test, p<0.05). Immunohistochemical staining showed ELF4 expression in the nuclei of PAAD cells, and GSEA showed a positive correlation between increased ELF4 expression and the cell cycle-related gene sets and PI3K/AKT/mTOR-related gene sets. Conclusions: The current result of the NET-CAGE enhancer analysis revealed that the KLF and ELF families might contribute to activating pancreatic cancer. As we know that KLF4 induces the pluripotency of somatic cells, KLF and ELF are considered to be cancer stemness-related genes. We are focusing on deciphering the regulatory element of the KLF and ELF to orchestrate downstream molecules of cancer stemness-related genes, which will lead to the intractability of pancreatic cancer. Citation Format: Tadashi Abe, Hideyuki Saito, Yusuke Nakano, Masahiro Hashimoto, Yuki Ando, Yuichi Hisamatsu, Takeo Toshima, Yusuke Yonemura, Takaaki Masuda, Ken Eto, Toru Ikegami, Koshi Mimori. Identification of the critical active enhancer and regulated transcripts contributing to the refractory nature of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1484.

Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids.

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

Mir-98-5p regulates gluconeogenesis and lipogenesis by targeting PPP1R15B in hepatocytes

Several reports suggest that circulatory miRNAs are deregulated in diverse diseases and used as markers for disease diagnosis and prognosis. Here we show that miR-98-5p, that is down-regulated in the circulation during diabetes, regulates hepatic gluconeogenesis and lipogenesis by targeting PPP1R15B. miR-98-5p overexpression significantly decreased the transcript and protein levels of PPP1R15B in hepatic HepG2 cells and increased p-eIF2α expression and these were prevented in the presence of its inhibitor. Two major hepatic hallmarks during diabetes i.e. hepatic lipid accumulation and glucose output were explored towards physiological relevance. As compared to scramble, overexpression of miR-98-5p decreased the transcript levels of both gluconeogenic and lipogenic genes together with a significant reduction in hepatic glucose production and fat accumulation in HepG2 cells. Using PASTAA to detect common transcription factors regulating these altered genes, CREB emerged as the most significantly enriched transcription factor. While miR-98-5p overexpression did not change the transcript levels of CREB, there was a significant change in its protein levels. While similar effects on gluconeogenic and lipogenic gene expression were detected using the PPP1R15B siRNA, the opposite was observed in the presence of miR-98-5p inhibitor alone. All these suggest that by targeting PPP1R15B, miR-98-5p regulates hepatic steatosis and glucose output; deregulation of which are characteristic hepatic features during diabetes. Therapeutic intervention of the miR-98/PPP1R15B axis might offer a potential strategy to target aberrant hepatic metabolism during diabetes.Graphical

Abstract PD10-03: PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC)

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort (SABCS19-GS2-02). Cell migration was assessed by the wound-healing assay. Results: We identified KLF4 among the top enriched TF binding motifs at the shared SEs in FOXA1-overexpressing MCF7-P cells and the FOXA1-amplified TamR cells. Analysis of our prior RNA-seq data of MCF7-P and TamR cells upon OE or siRNA KD of FOXA1, FRA1, or c-JUN (PMIDs 27791031, 31826955, 32424275, SABCS21-PD1-05) revealed KLF4, the Yamanaka factor for induced pluripotent stem cells, as a common target activated by the FOXA1/FRA1/c-JUN axis. We next identified 44 genes commonly down-regulated upon KLF4 KD in the MCF7-TamR cells. This KLF4-dependent 44-gene set was enriched in biological processes of embryonic development and tumor progression, preferentially dependent on ER in MCF7-TamR vs. P cells, highly elevated in ER+ metastases vs. primary tumors, and associated with poor outcome in ER+ BC treated with endocrine therapy. KLF4 KD, using the 2 siRNAs that generated similar pathway perturbations in MCF7-TamR cells, reduced TamR cell migration. Notably, among the genes co-dependent on KLF4 and ER in TamR cells, PYGB was the only gene with a PTR residing in an ER-bound SE established in TamR but not P cells. Glycogen phosphorylase B, encoded by PYGB, is the rate-limiting enzyme in glycogen degradation and plays a role in the progression of various tumors. Expression of KLF4, FOXA1, and FRA1 are commonly activated during the differentiation of human embryonic stem cells into foregut endoderm and in the inner core of fibroblasts of first-trimester human placenta villi, suggesting a unique role of KLF4 in mediating lineage-specific CRC, possibly by engaging PYGB and the glycogen metabolic pathway in advanced ER+ disease. Conclusions: Using SE-oriented integrative bioinformatics, we identified KLF4 as a potential novel target in the FOXA1/AP-1 transcriptional axis. As KLF4 binding motif resides in the unique ER-bound SEs of TamR cells, KLF4 likely forms an auto-regulated loop amplifying CRC in transcriptional reprogramming, among which the PYGB/glycogen metabolic pathway merits further investigation in endocrine-resistant ER+ disease. Citation Format: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff. PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-03.

Dissecting the effect of ethylene in the transcriptional regulation of chilling treatment in grapevine leaves

Ethylene (ETH) plays important roles in various development programs and stress responses in plants. In grapevines, ETH increased dramatically under chilling stress and is known to positively regulate cold tolerance. However, the role of ETH in transcriptional regulation during chilling stress of grapevine leaves is still not clear. To address this gap, targeted hormone profiling and transcriptomic analysis were performed on leaves of Vitis amurensis under chilling stress with and without aminoethoxyvinylglycine (AVG, a inhibitor of ETH synthesis) treatment. APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) and WRKY transcription factors (TF) were only the two highly enriched TF families that were consistently up-regulated during chilling stress but inhibited by AVG. The comparison of leaf transcriptomes between chilling treatment and chilling with AVG allowed the identification of potential ETH-regulated genes. Potential genes that are positively regulated by ETH are enriched in solute transport, protein biosynthesis, phytohormone action, antioxidant and carbohydrate metabolism. Conversely, genes related to the synthesis and signaling of ETH, indole-3-acetic acid (IAA), abscisic acid (ABA) were up-regulated by chilling treatment but inhibited by AVG. The contents of ETH, ABA and IAA also paralleled with the transcriptome data, which suggests that the response of ABA and IAA during chilling stress may regulate by ETH signaling, and together may belong to an integrated network of hormonal signaling pathways underpinning chilling stress response in grapevine leaves. Together, these findings provide new clues for further studying the complex regulatory mechanism of ETH under low-temperature stress in plants more generally and new opportunities for breeding cold-resilient grapevines.

HSSB1 (NABP2/OBFC2B) modulates the DNA damage and androgen-induced transcriptional response in prostate cancer.

Activation and regulation of androgen receptor (AR) signaling and the DNA damage response impact the prostate cancer (PCa) treatment modalities of androgen deprivation therapy (ADT) and radiotherapy. Here, we have evaluated a role for human single-strand binding protein 1 (hSSB1/NABP2) in modulation of the cellular response to androgens and ionizing radiation (IR). hSSB1 has defined roles in transcription and maintenance of genome stability, yet little is known about this protein in PCa. We correlated hSSB1 with measures of genomic instability across available PCa cases from The Cancer Genome Atlas (TCGA). Microarray and subsequent pathway and transcription factor enrichment analysis were performed on LNCaP and DU145 prostate cancer cells. Our data demonstrate that hSSB1 expression in PCa correlates with measures of genomic instability including multigene signatures and genomic scars that are reflective of defects in the repair of DNA double-strand breaks via homologous recombination. In response to IR-induced DNA damage, we demonstrate that hSSB1 regulates cellular pathways that control cell cycle progression and the associated checkpoints. In keeping with a role for hSSB1 in transcription, our analysis revealed that hSSB1 negatively modulates p53 and RNA polymerase II transcription in PCa. Of relevance to PCa pathology, our findings highlight a transcriptional role for hSSB1 in regulating the androgen response. We identified that AR function is predicted to be impacted by hSSB1 depletion, whereby this protein is required to modulate AR gene activity in PCa. Our findings point to a key role for hSSB1 in mediating the cellular response to androgen and DNA damage via modulation of transcription. Exploiting hSSB1 in PCa might yield benefits as a strategy to ensure a durable response to ADT and/or radiotherapy and improved patient outcomes.