Regulation Of Downstream Genes Research Articles

Targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for malate-aspartate shuttle and tumour progression.

A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown. Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test. c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients. These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.

Identification of mulberry leaf flavonoids and evaluating their protective effects on H2O2-induced oxidative damage in equine skeletal muscle satellite cells.

Introduction: Horses are susceptible to oxidative stress during strenuous endurance exercise, leading to muscle fatigue and damage. Mulberry leaf flavonoids (MLFs) possess significant antioxidant properties. However, the antioxidant efficacy of MLFs can be influenced by the extraction process, and their impact on H2O2-induced oxidative stress in equine skeletal muscle satellite cells (ESMCs) remains unexplored. Methods: Our study employed three extraction methods to obtain MLFs: ultrasound-assisted extraction (CEP), purification with AB-8 macroporous resin (RP), and n-butanol extraction (NB-EP). We assessed the protective effects of these MLFs on H2O2-induced oxidative stress in ESMCs and analyzed the MLF components using metabolomics. Results: The results revealed that pre-treatment with MLFs dose-dependently protected ESMCs against H2O2-induced oxidative stress. The most effective concentrations were 0.8mg/mL of CEP, 0.6mg/mL of RP, and 0.6mg/mL of NB-EP, significantly enhancing EMSC viability (p < 0.05). These optimized MLF concentrations promoted the GSH-Px, SOD and T-AOC activities (p < 0.05), while reducing MDA production (p < 0.05) in H2O2-induced ESMCs. Furthermore, these MLFs enhanced the gene expression, including Nrf2 and its downstream regulatory genes (TrxR1, GPX1, GPX3, SOD1, and SOD2) (p < 0.05). In terms of mitochondrial function, ESMCs pre-treated with MLFs exhibited higher basal respiration, spare respiratory capacity, maximal respiration, ATP-linked respiration compared to H2O2-induced ESMCs (p < 0.05). Additionally, MLFs enhanced cellular basal glycolysis, glycolytic reserve, and maximal glycolytic capacity (p < 0.05). Metabolomics analysis results revealed significant differences in mulberrin, kaempferol 3-O-glucoside [X-Mal], neohesperidin, dihydrokaempferol, and isobavachalcone among the three extraction processes (p < 0.05). Discussion: Our study revealed that MLFs enhance antioxidant enzyme activity, alleviate oxidative damage in ESMCs through the activation of the Nrf2 pathway, and improve mitochondrial respiration and cell energy metabolism. Additionally, we identified five potential antioxidant flavonoid compounds, suggesting their potential incorporation into the equine diet as a strategy to alleviate exercise-induced oxidative stress.

Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer.

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Transcriptional and post-translational regulation of MITF mediated by bHLH domain during the melanogenesis and melanocyte proliferation in Crassostrea gigas

Melanocyte differentiation is orchestrated by the master regulator transcription factor MITF. However, its ability to discern distinct binding sites linked to effective gene regulation remains poorly understood. This study aims to assess how co-activator acetyltransferase interacts with MITF to modulate their related lysine action, thereby mediating downstream gene regulation, including DNA affinity, stability, transcriptional activity, particularly in the process of shell pigmentation. Here, we have demonstrated that the CgMITF protein can be acetylated, further enabling selective amplification of the melanocyte maturation program. Collaboration with transcriptional co-regulator p300 advances MITF dynamically interplay with downstream targeted gene promoters. We have established that MITF activation was partially dependent on the bHLH domain, which was well conserved across species. The bHLH domain contained conserved lysine residues, including K6 and K43, which interacted with the E-box motif of downstream targeted-genes. Mutations at K6 and K43 lead to a decrease in the binding affinity of the E-box motif. CgMITF protein bound to the E-box motif within the promoter regions of the tyrosinase-related genes, contributing to melanogenesis, and also interacted with the E-box motif within the TBX2 promoter regions, associated with melanocyte proliferation. We elucidated how the bHLH domain links the transcriptional regulation and acetylation modifications in the melanocyte development in C. gigas.

Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy invivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil invitro and invivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

Abstract 2637: Super-enhancer-related m6A modification promotes immune escape in hepatocellular carcinoma associated with metabolic dysfunction-associated fatty liver disease via CCL4 signaling pathway inhibition

Abstract The incidence and mortality rates of hepatocellular carcinoma caused by metabolic dysfunction-associated fatty liver disease (MAFLD) have been increasing in recent decades, emphasizing the urgent need for new therapeutic approaches. Emerging evidence suggests that approximately 80% of super-enhancer RNAs (seRNAs) undergo selective m6A modifications. seRNA and m6A modifications have been implicated in the progression of various cancers, making them potential targets for therapeutic intervention. Based on our previous research, we hypothesize that the lack of m6A modification on the super-enhancer of CCL4 may contribute to the reduced expression of the CCL4 gene in HCC.To validate our hypothesis, we employ nano-chromatin immunoprecipitation combined with high-throughput sequencing (nano-ChIP-seq), m6A-seq (m6A-seq), single-cell RNA sequencing (scRNA-seq) on multiple models including clinical samples, cell lines, and mouse models. Interestingly, we have made a significant discovery that overexpression of CCL4 in tumor cells enhances the infiltration of CD8+ T cells, which are crucial for anti-tumor immune responses. In contrast, normal tissue cells undergo m6A modification on the CCL4 seRNAs, while this modification is notably absent in cancerous tissue. Consequently, the reduced expression of CCL4 could impair the tumor-killing capabilities of CD8+ T cells and facilitate immune evasion by the tumor.Furthermore, our results shed light on the underlying molecular mechanisms involving super-enhancers and m6A modifications in the regulation of immune escape in MAFLD HCC. We identify the specific super-enhancer and downstream regulatory genes influenced by m6A modification in MAFLD-associated HCC, providing valuable insights and novel targets for the treatment of this condition. Citation Format: Liangliang Xu. Super-enhancer-related m6A modification promotes immune escape in hepatocellular carcinoma associated with metabolic dysfunction-associated fatty liver disease via CCL4 signaling pathway inhibition [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 2637.

Identification and functional study of a novel variant of PAX9 causing tooth agenesis.

To search for pathogenic gene of a family with non-syndromic tooth agenesis, and explore the possible pathogenesis. A Chinese family with non-syndromic tooth agenesis was recruited and screened for the pathogenic variants by whole exome sequencing technology and co-segregation analysis. The subcellular localization of wild-type and mutant protein was detected by immunofluorescence assay. Cycloheximide chase assay was performed to examine the difference in degradation rate between mutant protein and wild-type one. Dual-luciferase reporter assays were conducted to explore the alterations of mutant protein in the regulation of downstream target genes. A novel missense variant of PAX9 (c.296C>A:p.A99D) was found in this family. Bioinformatics software showed β-return and the random coil were shortened in the p.A99D. The variant did not affect the subcellular localization of PAX9, but the degradation rate of p.A99D was accelerated (p < 0.05). p.A99D inhibited the activation of downstream target gene BMP4 (p < 0.05). This novel variant expands the pathogenic gene spectrum. The variant impaired the protein structure, accelerated the degradation of protein, and inhibited the activation of the downstream target gene BMP4, an upstream molecule in the TGF-β/BMP pathway, which may contribute to tooth agenesis in this family.

(058) Evaluation of Pathway Expression Following Down-Regulation of NELL1, a Potential Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we report on pathway analysis following up- and down-regulation of NELL1 using CRISPR modification in an in vitro setting. Objective This study aimed to investigate the influence of NELL1 modification on downstream gene regulation, focusing on pathways that were significantly regulated following NELL1 down-regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. Gene regulation was assessed using qRT-PCR. Results For down-regulation of NELL1, PCR analysis confirmed a 95% reduction in expression. Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 5,000 differentially expressed genes between, with significant changes in multiple pathways, including the HIF-1α pathway, which promotes keloid development by activating the TGF-β/Smad pathways. Additionally, we identified pathways related to ECM-receptor interaction, inflammation, apoptosis, and cell cycle regulation that are mediated by TGF-β/Smad signaling, which plays a crucial role in fibrosis. Our analysis also demonstrated upregulation of the PI3K-Akt pathway, which is involved in regulating type I collagen expression in response to TGF-β1. These results highlight the significance of NELL1 in fibrotic regulation and suggest its potential involvement in the development of PD. Conclusions We have created guides for and successfully down-regulated the expression of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. The identified modified pathways, including HIF-1α-mediated, TGF-β/Smad and PI3K-Akt, provide valuable insights into the potential molecular mechanisms underlying NELL1's role in regulation of fibrosis, and further investigation of these pathways may reveal potential therapeutic targets for fibrosis-related disorders. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Techfields Inc, Alto Neurosciences, Curebase.

SpecLoop predicts cell type-specific chromatin loop via transcription factor cooperation

Cell-type-Specific Chromatin Loops (CSCLs) are crucial for gene regulation and cell fate determination. However, the mechanisms governing their establishment remain elusive. Here, we present SpecLoop, a network regularization-based machine learning framework, to investigate the role of transcription factors (TFs) cooperation in CSCL formation. SpecLoop integrates multi-omics data, including gene expression, chromatin accessibility, sequence, protein-protein interaction, and TF binding motif data, to predict CSCLs and identify TF cooperations. Using high resolution Hi-C data as the gold standard, SpecLoop accurately predicts CSCL in GM12878, IMR90, HeLa–S3, K562, HUVEC, HMEC, and NHEK seven cell types, with the AUROC values ranging from 0.8645 to 0.9852 and AUPR values ranging from 0.8654 to 0.9734. Notably SpecLoop demonstrates improved accuracy in predicting long-distance CSCLs and identifies TF complexes with strong predictive ability. Our study systematically explores the TFs and TF pairs associated with CSCL through effective integration of diverse omics data. SpecLoop is freely available at https://github.com/AMSSwanglab/SpecLoop.

S1 basic leucine zipper transcription factors shape plant architecture by controlling C/N partitioning to apical and lateral organs

Plants tightly control growth of their lateral organs, which led to the concept of apical dominance. However, outgrowth of the dormant lateral primordia is sensitive to the plant's nutritional status, resulting in an immense plasticity in plant architecture. While the impact of hormonal regulation on apical dominance is well characterized, the prime importance of sugar signaling to unleash lateral organ formation has just recently emerged. Here, we aimed to identify transcriptional regulators, which control the trade-off between growth of apical versus lateral organs. Making use of locally inducible gain-of-function as well as single and higher-order loss-of-function approaches of the sugar-responsive S1-basic-leucine-zipper (S1-bZIP) transcription factors, we disclosed their largely redundant function in establishing apical growth dominance. Consistently, comprehensive phenotypical and analytical studies of S1-bZIP mutants show a clear shift of sugar and organic nitrogen (N) allocation from apical to lateral organs, coinciding with strong lateral organ outgrowth. Tissue-specific transcriptomics reveal specific clade III SWEET sugar transporters, crucial for long-distance sugar transport to apical sinks and the glutaminase GLUTAMINE AMIDO-TRANSFERASE 1_2.1, involved in N homeostasis, as direct S1-bZIP targets, linking the architectural and metabolic mutant phenotypes to downstream gene regulation. Based on these results, we propose that S1-bZIPs control carbohydrate (C) partitioning from source leaves to apical organs and tune systemic N supply to restrict lateral organ formation by C/N depletion. Knowledge of the underlying mechanisms controlling plant C/N partitioning is of pivotal importance for breeding strategies to generate plants with desired architectural and nutritional characteristics.

Transcription factor LBD16 targets cell wall modification/ion transport genes in peach lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.

MicroRNA-93-5p regulates odontogenic differentiation and dentin formation via KDM6B

BackgroundEpigenetic factors influence the odontogenic differentiation of dental pulp stem cells and play indispensable roles during tooth development. Some microRNAs can epigenetically regulate other epigenetic factors like DNA methyltransferases and histone modification enzymes, functioning as epigenetic-microRNAs. In our previous study, microarray analysis suggested microRNA-93-5p (miR-93-5p) was differentially expressed during the bell stage in human tooth germ. Prediction tools indicated that miR-93-5p may target lysine-specific demethylase 6B (KDM6B). Therefore, we explored the role of miR-93-5p as an epi-miRNA in tooth development and further investigated the underlying mechanisms of miR-93-5p in regulating odontogenic differentiation and dentin formation.MethodsThe expression pattern of miR-93-5p and KDM6B of dental pulp stem cells (DPSCs) was examined during tooth development and odontogenic differentiation. Dual luciferase reporter and ChIP-qPCR assay were used to validate the target and downstream regulatory genes of miR-93-5p in human DPSCs (hDPSCs). Histological analyses and qPCR assays were conducted for investigating the effects of miR-93-5p mimic and inhibitor on odontogenic differentiation of hDPSCs. A pulpotomy rat model was further established, microCT and histological analyses were performed to explore the effects of KDM6B-overexpression and miR-93-5p inhibition on the formation of tertiary dentin.ResultsThe expression level of miR-93-5p decreased as odontoblast differentiated, in parallel with elevated expression of histone demethylase KDM6B. In hDPSCs, miR-93-5p overexpression inhibited the odontogenic differentiation and vice versa. MiR-93-5p targeted 3′ untranslated region (UTR) of KDM6B, thereby inhibiting its protein translation. Furthermore, KDM6B bound the promoter region of BMP2 to demethylate H3K27me3 marks and thus upregulated BMP2 transcription. In the rat pulpotomy model, KDM6B-overexpression or miR-93-5p inhibition suppressed H3K27me3 level in DPSCs and consequently promoted the formation of tertiary dentin.ConclusionsMiR-93-5p targets epigenetic regulator KDM6B and regulates H3K27me3 marks on BMP2 promoters, thus modulating the odontogenic differentiation of DPSCs and dentin formation.

PI3Kδ Inhibition Potentiates Glucocorticoids in B-lymphoblastic Leukemia by Decreasing Receptor Phosphorylation and Enhancing Gene Regulation.

Glucocorticoids are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency should improve treatment. We previously showed that inhibition of the lymphoid-restricted PI3Kδ with idelalisib enhances glucocorticoid activity in B-ALL cells. Here, we show that idelalisib enhances glucocorticoid potency in 90% of primary B-ALL specimens and is most pronounced at sub-saturating doses of glucocorticoids near the EC50. Potentiation is associated with enhanced regulation of all glucocorticoid-regulated genes, including genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at PI3Kδ/MAPK1 (ERK2) targets S203 and S226. Ablation of these phospho-acceptor sites enhances sensitivity to glucocorticoids with ablation of S226 in particular reducing synergy. We also show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro. We propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. This mechanism and the response of patient specimens suggest that idelalisib will benefit most patients with B-ALL, but particularly patients with less responsive, including high-risk, disease. This combination is also promising for the development of less toxic glucocorticoid-sparing therapies.

METTL14-mediated lncRNA XIST silencing alleviates GDM progression by facilitating trophoblast cell proliferation and migration via the miR-497-5p/FOXO1 axis.

Gestational diabetes mellitus (GDM),a prevalent complication during the gestation period, has been linked to impaired proliferation and migration of trophoblasts causing placental maldevelopment. We previously found that lncRNA X-inactive specific transcript (XIST)played an essential role in GDM progression. Here, we investigated the precise biological functions as well as the upstream and downstream regulatory mechanisms of XIST in GDM. We found that XISTand forkheadbox O1 (FOXO1) were conspicuously upregulated and miR-497-5p and methyltransferase-like 14 (METTL14) were downregulated in the placentas of GDM patients. XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells. METTL14 inhibited XIST expression through m6A methylation modification. XIST overexpression abrogated the positive effect of METTL14 overexpression on HG-cultured HTR8/SVneo cell progression. MiR-497-5p and FOXO1 are downstream regulatory genes of XIST in HTR8/SVneo cells. Reverse experiments illustrated that XIST mediated HTR8/SVneo cell functions by regulating the miR-497-5p/FOXO1 axis. Additionally, XIST silencing augmented glucose tolerance and alleviated fetal detrimental changes in GDM rats. To conclude, METTL14-mediated XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells via the miR-497-5p/FOXO1 axis, thereby alleviating GDM progression in rats.

Large-scale loss-of-function perturbations reveal a comprehensive epigenetic regulatory network in breast cancer.

Epigenetic abnormalities have a critical role in breast cancer by regulating gene expression; however, the intricate interrelationships and key roles of approximately 400 epigenetic regulators in breast cancer remain elusive. It is important to decipher the comprehensive epigenetic regulatory network in breast cancer cells to identify master epigenetic regulators and potential therapeutic targets. We employed high-throughput sequencing-based high-throughput screening (HTS2) to effectively detect changes in the expression of 2,986 genes following the knockdown of 400 epigenetic regulators. Then, bioinformatics analysis tools were used for the resulting gene expression signatures to investigate the epigenetic regulations in breast cancer. Utilizing these gene expression signatures, we classified the epigenetic regulators into five distinct clusters, each characterized by specific functions. We discovered functional similarities between BAZ2B and SETMAR, as well as CLOCK and CBX3. Moreover, we observed that CLOCK functions in a manner opposite to that of HDAC8 in downstream gene regulation. Notably, we constructed an epigenetic regulatory network based on the gene expression signatures, which revealed 8 distinct modules and identified 10 master epigenetic regulators in breast cancer. Our work deciphered the extensive regulation among hundreds of epigenetic regulators. The identification of 10 master epigenetic regulators offers promising therapeutic targets for breast cancer treatment.

Identifying LiaFSR Residues Contributing to ExPortal Integrity and Response to Antimicrobials in Group A Streptococcus

Abstract Background Group A Streptococcus (GAS), or Streptococcus pyogenes, is responsible for a myriad of diseases (pharyngitis, scarlet fever, impetigo, toxic shock syndrome) of which several are prevalent in the pediatric population. The LiaFSR two-component regulatory system (TCS) and the ExPortal functional membrane microdomain (FMM) of GAS have been shown to coordinate the response to cell membrane stress when the bacterium is exposed to antimicrobials or antimicrobial peptides (AMPs). The ExPortal coordinates translocation and processing of secreted GAS proteins and contributes to sensing membrane. In an unstressed state, LiaF acts as an inhibitor of LiaS activity, both residing within the ExPortal. In a stressed state, such as exposure to antimicrobial agents, LiaF and S dissociate, allowing LiaS to phosphorylate LiaR, resulting in subsequent activation and expression of downstream target genes. Genomic analysis indicates a strict conservation of specific protein domains and certain amino acid residues in LiaF, S, and R, which suggests their critical involvement in the LiaFSR mechanism of action. We hypothesize that conserved amino acid residues in LiaFSR, such as the LiaF C-terminal domain (CTD) and a 3xK residue motif, are essential to ExPortal formation and maintenance and may contribute to differences in antimicrobial susceptibility and virulence. Methods Two different mutant strains were analyzed and compared to (wild-type) WT: one lacking the LiaF CTD (LiaF_∆CTD) and one mutagenized in the 3xK motif (LiaF_K91-93A). Five different assays were used to examine phenotypes associated with LiaFSR activity: SpeB protease activity through bacterial growth on milk plates, ExPortal integrity via fluorescence microscopy, diamide-induced oxidative stress tolerance, antimicrobial/antimicrobial peptide (AM/AMP) susceptibility, and a direct bactericidal assay in human blood (Lancefield). For the AM/AMP susceptibility, we tested 6 different antimicrobial agents with varying mechanisms of action: bacitracin, polymyxin B, LL-37, nisin, daptomycin, and HNP-1. Results Milk plate analysis revealed a significant decrease in SpeB production in the LiaF-∆CTD vs. WT, but no difference in LiaF-K91-93A relative to WT. During fluorescence microscopy, no significant differences were detected between foci of fluorescence of either mutant strain relative to WT. When exposed to diamide-induced oxidative stress, both mutants exhibited increased tolerance relative to WT. AM/AMP susceptibility assays did not reveal any significant differences for any antimicrobial agent between either mutant strain vs. WT. Finally, the Lancefield assay revealed a significant decrease in survival in human blood between both mutant strains and WT. Mutant strains also had significantly decreased survival relative to a strain lacking LiaF (∆LiaF). Conclusions The results of the AM/AMP susceptibility assay suggest that, unlike the LiaFSR system in Enterococcus, LiaF function does not contribute to LiaFSR-dependent susceptibility. Significant differences in survival in blood indicate that the LiaF residues studied are physiologically relevant in GAS infection. However, their exact mechanism underlying this contribution is being investigated. Future research will examine downstream gene regulation to elucidate the LiaFSR-associated mechanism of decreased survival in human blood, with the aim of defining determinants of GAS bacteremia.

The ftz upstream element drives late ftz stripes but is not required for regulation of ftz target genes

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz striped expression, with all stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells

HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339–39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.

Transcription factor TEAD4 facilitates glycolysis and proliferation of gastric cancer cells by activating PKMYT1

BackgroundGastric cancer (GC) ranks third for cancer deaths worldwide, and glycolysis is a hallmark of several cancers, including GC. TEAD4 plays a role in establishing an oncogenic cascade in cancers, including GC. Whether TEAD4 can influence the glycolysis of GC cells remains uncovered. Hence, this study attempted to investigate the impact on glycolysis of GC cells by TEAD4. MethodsBy using bioinformatics analysis, differentially expressed mRNAs were screened, and downstream regulatory genes were predicted. Expression levels of TEAD4 and PKMYT1 were assessed by qRT-PCR. The binding sites between TEAD4 and PKMYT1 were predicted by the JASPAR database, meanwhile their modulatory relationship was confirmed through dual-luciferase assay and chromatin Immunoprecipitation (ChIP). Cell viability and proliferation were assayed via CCK-8 and colony formation assays. Glycolysis was measured by assaying extracellular acidification rate, oxygen consumption rate, and production of pyruvic acid, lactate, citrate, and malate. Expression levels of proteins (HK-2 and PKM2) related to glycolysis were assessed by Western blot. ResultsTEAD4 was upregulated in GC tissues and cells. TEAD4 knockdown substantially repressed glycolysis and proliferation of GC cells. PKMYT1, the target gene downstream of TEAD4, was identified via bioinformatics prediction, and its expression was elevated in GC. Dual-luciferase and ChIP assay validated the targeted relationship between the promoter region of PKMYT1 and TEAD4. As revealed by rescue experiments, the knockdown of TEAD4 reversed the stimulative effect on GC cell glycolysis and proliferation by forced expression of PKMYT1. ConclusionTEAD4 activated PKMYT1 to facilitate the proliferation and glycolysis of GC cells. TEAD4 and PKMYT1 may be possible therapeutic targets for GC.

A Neanderthal/Denisovan GLI3 variant contributes to anatomical variations in mice.

Changes in genomic structures underlie phenotypic diversification in organisms. Amino acid-changing mutations affect pleiotropic functions of proteins, although little is known about how mutated proteins are adapted in existing developmental programs. Here we investigate the biological effects of a variant of the GLI3 transcription factor (GLI3R1537C) carried in Neanderthals and Denisovans, which are extinct hominins close to modern humans. R1537C does not compromise protein stability or GLI3 activator-dependent transcriptional activities. In contrast, R1537C affects the regulation of downstream target genes associated with developmental processes. Furthermore, genome-edited mice carrying the Neanderthal/Denisovan GLI3 mutation exhibited various alterations in skeletal morphology. Our data suggest that an extinct hominin-type GLI3 contributes to species-specific anatomical variations, which were tolerated by relaxed constraint in developmental programs during human evolution.