Transcriptional Regulatory Proteins Research Articles

Neuronal RNA processing: cross-talk between transcriptional regulation and RNA-binding proteins

Neuronal RNA processing: cross-talk between transcriptional regulation and RNA-binding proteins

Evaluating the potential impact of sodium-glucose cotransporter-2 inhibitor "canagliflozin" on the hepatic damage triggered by hypertension in rats.

Hypertension (HTN) is a prevalent chronic disease. HTN and liver disease association is extensively noted. Thus, finding a medication that can alleviate HTN and its accompanying liver insult would be promising. This study investigated the potential impacts of canagliflozin "sodium-glucose cotransporter-2 inhibitor" on the liver of the Nω-nitro-L-arginine methyl ester (L-NAME)-induced HTN rat model. Twenty-four adult male rats were divided into four groups; negative control group, canagliflozin group, L-NAME group: 50 mg/kg of L-NAME was injected daily for 5 weeks and L-NAME + canagliflozin group: 1 week after L-NAME injection both L-NAME + canagliflozin (40 mg/kg) were given concomitantly daily for further 4 weeks. Liver functions, serum lipid profile, hepatic oxidative/nitrative stress biomarkers, gene expression of lipogenic enzymes, B-cell lymphoma 2 (Bcl2), and DNA fragmentation, were measured. Besides, hepatic histology and immunohistochemistry of nuclear factor kappa B (NF-κB) and endothelial nitric oxide synthase (eNOS) were assessed. Canagliflozin improved hepatic lipogenesis via the downregulation of fatty acid synthase (FAS) and transcriptional regulatory element binding protein 1c (SREBP1c) genes leading to an improved serum lipid profile. Further, canagliflozin modified the eNOS/inducible nitric oxide synthase (iNOS) pathway and decreased the NF-κB immunoreactivity besides restoring the oxidants-antioxidants balance; increased reduced glutathione concomitant with declined malondialdehyde. This improvement of the liver was mirrored by the significant restoration of liver architecture and confirmed by the preserved liver DNA content and upregulation of the antiapoptotic Bcl2 mRNA level and attenuation of the alanine transaminase, aspartate aminotransferase. In conclusion, canagliflozin is a promising anti-hypertensive and hepatic-supportive medication. RESEARCH HIGHLIGHTS: Canagliflozin's antioxidant, anti-inflammatory, anti-lipogenic, and antiapoptotic characteristics mitigate remote liver compromise caused by hypertension. Canagliflozin can be exploited as a hepatoprotective and antihypertensive medication.

The Role of Long Non-Coding RNF144A-AS1 in Cancer Progression.

RNAs transcribing more than 200 nucleotides without encoding proteins are termed long non-coding RNAs (LncRNAs). LncRNAs can be used as decoy molecules, signal molecules, scaffolds, and guide molecules. Long non-coding RNAs can interact with DNA, chromatin-modifying complexes, and transcriptional regulatory proteins, regulating gene expression in the cell nucleus. It is distributed in cytoplasm; they also participate in mRNA degradation and translational regulation via miRNAs, other transcription products, and proteins. They play a significant role in the development of various diseases, including tumors. Cancer seriously threatens human life and health. Regretfully, a great deal of newly diagnosed cancer patients found to have metastasized. RNF144A-AS1, also referred to as GRASLND, was initially recognized for its regulation of chondrogenic differentiation in MSCs. Focusing on RNF144A-AS1, this review summarizes and discusses the latest progress of RNF144A-AS1 in bladder cancer, glioblastoma, papillary renal cell carcinoma, gastric cancer, osteosarcoma, head and neck squamous cell carcinoma, and ovarian cancer. RNF144A-AS1 has good potential in tumor treatment and diagnosis.

Analytical Post-Embedding Immunogold–Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold–electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

Analytical Post-Embedding Immunogold-Electron Microscopy with Direct Gold-Labelled Monoclonal Primary Antibodies against RIBEYE A- and B-Domain Suggests a Refined Model of Synaptic Ribbon Assembly.

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

The viable but non-culturable (VBNC) status of Shewanella putrefaciens (S. putrefaciens) with thermosonication (TS) treatment

Although thermosonication (TS) treatment has been widely used in food sterilization, the viable but non‐culturable (VBNC) of bacteria with TS treatment has still concerned potential food safety and public health. The molecular mechanism of VBNC status of bacteria with TS treatment is not clearly known. Therefore, in this study, we used Shewanella putrefaciens, which was a common putrefactive bacteria in aquatic products, to study the VBNC state of bacteria with TS treatment. Firstly, our results revealed that S. putrefaciens still could enter the VBNC state after TS treatments: 50 kHz, 300 W, 30 min ultrasonic treatment and 70 °C heating; Subsequently, we found the VBNC state of S. putrefaciens can resist the damage of TS treatment, such as cell wall break, DNA degradation, etc; Finally, four-dimensional data-independent acquisition-based proteomics showed that under VBNC state, S. putrefaciens upregulated functional proteins to resist TS treatment, such as: ribosomal proteins to accelerate the synthesis of stress proteins to counteract TS treatments, ornithine decarboxylase SpeF and MraY to repair TS treatment-induced damage, etc. Meanwhile, S. putrefaciens downregulates metabolic and transport functional proteins such as dehydrogenase to reduce the metabolism. Importantly, among those proteins, the ribosomal transcriptional regulatory protein family, such as rpsB, etc, may be the key proteins for S. putrefaciens entering VBNC state. This finding can provide some new strategies for preventing VBNC status of bacteria with TS treatment, such as: inhibition of key proteins, etc.

Construction of a Multicellular Communication Network Model for Cell Co-Culture Technology and Evaluation of Its Simulation Capability

Abstract Objective: Cell co-culture technology has been widely used to analyze the effects of drugs on cell proliferation and the expression of some proteins in cells, especially in the field of traditional Chinese medicine (TCM); however, the interactions between cells and the transmission of TCM effects between cells have not been adequately studied. Materials and Methods: Using data on gene transcription regulation, biological response, signal channel, and cell-specific expression protein, we built a network for cell types based on entity grammar. Through the correspondence and location information of signal molecules and receptors, type-specific networks of single cells were connected and a multicellular network of smooth muscle cells, neurons, and vascular endothelial cells was constructed. The mechanism of action of nimodipine was analyzed based on the multicellular communication network and its simulation capability was evaluated. Results: The outputs generated by the model developed in this study showed that nimodipine inhibited smooth muscle contraction, due to the overload of Ca2+ and the toxicity of excitatory amino acids, and protected neurons and vascular endothelial cells by supporting cell proliferation and inhibiting cell apoptosis. These results were consistent with the known mechanism of nimodipine action, thus confirming that the multicellular network can be used to study the transmission of drug effects among cells. Conclusions: This study lays a foundation for the analysis of the transmission of drug effects in multi-cells, tissues, organs, and other spatial scales through multicellular co-culture experiments, based on a multicellular communication network. In addition, it provides a biological network model for the analysis of TCM action mechanisms.

Regulation of eukaryotic transcription initiation in response to cellular stress

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

Comprehensive analysis of KLF family reveals KLF6 as a promising prognostic and immune biomarker in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors worldwide, with extremely aggressive and complicated biology. Krüppel-like factors (KLFs) encode a series of transcriptional regulatory proteins and play crucial roles in a variety of processes, including tumor cell differentiation and proliferation. However, the potential biological functions and possible pathways of KLFs in the progression of PDAC remain elusive.MethodsWe systematically evaluated the transcriptional variations and expression patterns of KLFs in pancreatic cancer from the UCSC Xena. Based on difference analysis, the non-negative matrix factorization (NMF) algorithm was utilized to identify the immune characteristics and clinical significance of two different subtypes. The multivariate Cox regression was used to construct the risk model and then explore the differences in tumor immune microenvironment (TIME) and drug sensitivity between high and low groups. Through single-cell RNA sequencing (scRNA-seq) analysis, we screened KLF6 and further investigated its biological functions in pancreatic cancer and pan-cancer.ResultsThe KLFs exhibited differential expression and mutations in the transcriptomic profile of PDAC. According to the expression of KLFs, patients were classified into two distinct subtypes, each exhibiting significant differences in prognosis and TIME. Moreover, the KLF signature was developed using univariate Cox and Lasso regression, which proved to be a reliable and effective prognostic model. Furthermore, the KLF_Score was closely associated with immune infiltration, response to immunotherapy, and drug sensitivity and we screened small molecule compounds targeting prognostic genes separately. Through scRNA-seq analysis, KLF6 was selected to further demonstrate its role in the malignance of PC in vitro. Finally, pan-cancer analysis emphasized the biological significance of KLF6 in multiple types of tumors and its clinical utility in assessing cancer prognosis.ConclusionThis study elucidated the pivotal role of KLF family genes in the malignant development of PC through comprehensive analysis and revealed that KLF6 would be a novel diagnostic biomolecule marker and potential therapeutic target for PDAC.

Human T-cell lymphotropic virus type 1 (HTLV-1) grip on T-cells: investigating the viral tapestry of activation

IntroductionHuman T-cell Lymphotropic virus type 1 (HTLV-1) belongs to retroviridae which is connected to two major diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and Adult T-cell leukemia/lymphoma (ATLL). This study aims to investigate the mRNA expressions of key proteins correlated to T-cell activation in asymptomatic carriers (ACs) HTLV-1 infected patients, shedding light on early molecular events and T-cell activation following HTLV-1 infection.Material and MethodsThe study involved 40 participants, including 20 ACs and 20 healthy subjects. Blood samples were collected, ELISA assessment for screening and confirmation with PCR for Trans-activating transcriptional regulatory protein (Tax) and HTLV-1 basic leucine zipper factor (HBZ) of the HTLV-1 were done. mRNA expressions of C-terminal Src kinase (CSK), Glycogen Synthase Kinase-3 Beta (GSK3β), Mitogen-Activated Protein Kinase 14 (MAP3K14 or NIK), Phospholipase C Gamma-1 (PLCG1), Protein Tyrosine Phosphatase non-Receptor Type 6 (PTPN6) and Mitogen-Activated Protein Kinase Kinase Kinase-7 (SLP-76) and Mitogen-Activated Protein Kinase14 (MAP3K7 or TAK1) were assayed using RT-qPCR. Statistical analyses were performed using PRISM and SPSS software.ResultsWhile there were no significant upregulation in CSK and PTPN6 in ACs compared to healthy individuals, expression levels of GSK3β, MAP3K14, PLCG1, SLP-76, and TAK1 were significantly higher in ACs compared to healthy subjects which directly contributes to T-cell activation in the HTLV-1 ACs.ConclusionHTLV-1 infection induces differential mRNA expressions in key proteins associated with T-cell activation. mRNAs related to T-cell activation showed significant upregulation compared to PTPN6 and CSK which contributed to T-cell regulation. Understanding these early molecular events in ACs may provide potential markers for disease progression and identify therapeutic targets for controlling viral replication and mitigating associated diseases. The study contributes novel insights to the limited literature on T-cell activation and HTLV-1 pathogenesis.

Hedyotis diffusa Willd and Astragalus membranaceus May Exert Anti-colon Cancer Effects by Affecting AKTI Expression, as Determined by Network Pharmacology and Molecular Docking.

Network pharmacology is a novel approach that uses bioinformatics to predict multitarget drugs and ingredient-target interactions in various diseases. A thorough search of previously published studies revealed that Hedyotis diffusa Willd (HDW) and Astragalus membranaceus (AM) possess anticancer activity. Colon cancer (CC) is one of the most common malignant tumors of the digestive tract and occurs in the colon. Herein, we explored the effect of two drugs in the treatment of CC. The present study aimed to predict and verify the effect of these two drugs in the treatment of CC. To explore the molecular mechanisms of the "HDW-AM" drug in the treatment of CC, we analyzed its principal efficiency in terms of ingredients, target spots, and pathways via network pharmacology, molecular docking, and experimental verification. The ingredients and their gene target sites were searched and screened through the TCMSP platform according to specific filtering conditions. Subsequently, components corresponding to the gene targets were chosen to construct the drug component-target network. The GEO (Gene Expression Omnibus) dataset was used to collect and screen for gene chips under CC and normal conditions, obtain differential genes, and construct a volcano map. The intersection genes between drug and disease targets were screened, the ".tsv" file was downloaded from the STRING platform and imported into Cytoscape 3.8.0 for visualization, a protein-protein interaction (PPI) network was constructed, the core targets were identified, and the common components with core targets were docked through Autodock Tools-1.5.6. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out through the Metascape platform to determine the major pathways. The CCK-8 (Cell Counting Kit-8) assay verified the effect of AKT1 on cell proliferation after treatment with quercetin. After the screening, 3658 DEGs (1841 downregulated and 1817 upregulated) were obtained from the GSE75970 gene chip; 21 active components and 220 targets were identified from the drugs. Subsequently, ten core genes (including AKT1, P53, and CASP3) and six major components were screened. GO functional analysis and KEGG analysis revealed that "HDWAM" regulates cell migration and motility through the combination of a transcription regulator complex, membrane rafts, vesicle lumen, and protein kinases via the MAPK, PI3K-Akt, and IL17 signaling pathways. The molecular docking results suggested that quercetin binds to AKT1, TP53, TNF, and CASP3. HDW-AM may exert a therapeutic effect on CC by modulating AKT1, TP53, TNF, and CASP3 and through signaling pathways. A CCK-8 cytotoxicity assay verified that quercetin affects cell viability through AKT1. The current study provides a theoretical basis for an in-depth investigation into the molecular mechanism of the "HDW-AM" drug in CC treatment via network pharmacology, molecular docking, and experimental verification.

Hepatitis B virus X protein promotes tumor glycolysis by downregulating lncRNA OIP5-AS1/HKDC1 in HCC

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide, with Hepatitis B virus (HBV) infection being the leading cause. This study aims to investigate the role of HBV in HCC pathogenesis involving glucose metabolism. Long non-coding RNA (lncRNA) OIP5-AS1 was significantly downregulated in HBV-positive HCC patients, and its low expression indicated a poor prognosis. This lncRNA was primarily localized in the cytoplasm, acting as a tumor suppressor. HBV protein X (HBx) repressed OIP5-AS1 expression by inhibiting a ligand-activated transcriptional factor peroxisome proliferator-activated receptor α (PPARα). Furthermore, mechanistic studies revealed that OIP5-AS1 inhibited tumor growth by suppressing Hexokinase domain component 1 (HKDC1)-mediated glycolysis. The expression of HKDC1 could be enhanced by transcriptional factor sterol regulatory element-binding protein 1 (SREBP1). OIP5-AS1 facilitated the ubiquitination and degradation of SREBP1 to suppress HKDC1 transcription, which inhibited glycolysis. The results suggest that lncRNA OIP5-AS1 plays an anti-oncogenic role in HBV-positive HCC via the HBx/OIP5-AS1/HKDC1 axis, providing a promising diagnostic marker and therapeutic target for HBV-positive HCC patients.

TDP2 is a regulator of estrogen-responsive oncogene expression.

With its ligand estrogen, the estrogen receptor (ER) initiates a global transcriptional program, promoting cell growth. This process involves topoisomerase 2 (TOP2), a key protein in resolving topological issues during transcription by cleaving a DNA duplex, passing another duplex through the break, and repairing the break. Recent studies revealed the involvement of various DNA repair proteins in the repair of TOP2-induced breaks, suggesting potential alternative repair pathways in cases where TOP2 is halted after cleavage. However, the contribution of these proteins in ER-induced transcriptional regulation remains unclear. We investigated the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme for the removal of halted TOP2 from the DNA ends, in the estrogen-induced transcriptome using both targeted and global transcription analyses. MYC activation by estrogen, a TOP2-dependent and transient event, became prolonged in the absence of TDP2 in both TDP2-deficient cells and mice. Bulk and single-cell RNA-seq analyses defined MYC and CCND1 as oncogenes whose estrogen response is tightly regulated by TDP2. These results suggest that TDP2 may inherently participate in the repair of estrogen-induced breaks at specific genomic loci, exerting precise control over oncogenic gene expression.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Abstract 3874: Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues, with full pathway inhibition at 4 hours post treatment that was restored by 24 hours. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. To understand the dynamic response of malignant cells following RAS-GTP inhibition, we did single cell RNA sequencing on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms [Maroling., et al. 2006; Alvarez., et al. 2016], we performed regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of >5000 transcriptional regulatory proteins per cell. Additionally, we assessed the impact of treatment on malignant cell subtypes as previously identified by our team based on their sets of hyperactivated developmental transcription factors [Laise., et al. 2022]. As expected, RAS pathway transcription factors, such as FOS and JUN, were repressed relative to controls in malignant cells by 4 hours after treatment with RMC-7977. However, at 24 hours, responses varied by cellular subtype: well-differentiated Gastrointestinal Lineage State (GLS) cells had restored MAPK activity, while poorly differentiated Morphogenic State (MOS) cells were still inactivated. Strikingly, we found that after one week of treatment (in actively regressing tumors, representing a state of residual disease), nearly all malignant cells had assumed the GLS state. Acquisition of resistance after weeks or months of treatment was associated with a restored diversity of malignant cell states, suggesting that the GLS state serves as a regulatory “safe harbor” in which residual malignant cells can undergo evolution and adaptation. Mechanistically, we found that RAS pathway signaling directly activates transcriptional regulators that drive the MOS state, elucidating an early adaptive response to RAS-GTP inhibition that enables residual cells to survive treatment prior to development of acquired resistance. Citation Format: Lorenzo Tomassoni, Urszula N. Wasko, Alvaro C. Garcia, Tanner C. Dalton, Pasquale Laise, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP 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 3874.

Genome-wide identification of GATA transcription factors in tetraploid potato and expression analysis in differently colored potato flesh.

The GATA gene family belongs to a kind of transcriptional regulatory protein featuring a zinc finger motif, which is essential for plant growth and development. However, the identification of the GATA gene family in tetraploid potato is still not performed. In the present research, a total of 88 GATA genes in the tetraploid potato C88.v1 genome were identified by bioinformatics methods. These StGATA genes had an uneven distribution on 44 chromosomes, and the corresponding StGATA proteins were divided into four subfamilies (I-IV) based on phylogenetic analysis. The cis-elements of StGATA genes were identified, including multiple cis-elements related to light-responsive and hormone-responsive. The collinearity analysis indicates that segmental duplication is a key driving force for the expansion of GATA gene family in tetraploid potato, and that the GATA gene families of tetraploid potato and Arabidopsis share a closer evolutionary relationship than rice. The transcript profiling analysis showed that all 88 StGATA genes had tissue-specific expression, indicating that the StGATA gene family members participate in the development of multiple potato tissues. The RNA-seq analysis was also performed on the tuber flesh of two potato varieties with different color, and 18 differentially expressed GATA transcription factor genes were screened, of which eight genes were validated through qRT-PCR. In this study, we identified and characterized StGATA transcription factors in tetraploid potato for the first time, and screened differentially expressed genes in potato flesh with different color. It provides a theoretical basis for further understanding the StGATA gene family and its function in anthocyanin biosynthesis.

YAP1/TAZ Mediates Rumen Epithelial Cell Proliferation but Not Short-Chain Fatty Acid Metabolism In Vitro.

Promoting rumen development is closely related to the health and efficient growth of ruminants. The transcriptional co-activators Yes1-associated protein (YAP1) and WW domain-containing transcription regulator protein 1 (TAZ) are key regulators of the mammalian epithelium. In the present study, we assessed the impact of YAP1/TAZ on rumen epithelial (RE) cell proliferation using their activator GA-017 (GA) and inhibitor verteporfin (VP). We also investigated whether YAP1/TAZ-dependent alteration was involved in the RE developmental process induced by sodium butyrate (SB). The results indicated that GA promoted RE cell proliferation, while VP disrupted RE cell proliferation. The Hippo, Wnt, and calcium signaling pathways were altered following the regulation of YAP1/TAZ. Upon YAP1/TAZ activation, the expression of CCN1/2 increased. However, when YAP1/TAZ was inhibited, the expression of BIRC3 decreased. In the SB-treated cells, YAP1/TAZ-induced changes were not observed. SB increased the expressions of differentiated cell marker genes and genes involved in short-chain fatty acid (SCFA) metabolism, while YAP1/TAZ did not. Thus, YAP1/TAZ could be potential targets for regulating RE cell proliferation but not for SCFA metabolism. SB could not affect YAP1/TAZ. These findings broaden our understanding of the role of YAP1/TAZ and their regulators in RE development.

The role of transcriptional regulators in metal ion homeostasis of Mycobacterium tuberculosis.

Metal ions are essential trace elements for all living organisms and play critical catalytic, structural, and allosteric roles in many enzymes and transcription factors. Mycobacterium tuberculosis (MTB), as an intracellular pathogen, is usually found in host macrophages, where the bacterium can survive and replicate. One of the reasons why Tuberculosis (TB) is so difficult to eradicate is the continuous adaptation of its pathogen. It is capable of adapting to a wide range of harsh environmental stresses, including metal ion toxicity in the host macrophages. Altering the concentration of metal ions is the common host strategy to limit MTB replication and persistence. This review mainly focuses on transcriptional regulatory proteins in MTB that are involved in the regulation of metal ions such as iron, copper and zinc. The aim is to offer novel insights and strategies for screening targets for TB treatment, as well as for the development and design of new therapeutic interventions.

Comparative genomics reveals distinct diversification patterns among LysR-type transcriptional regulators in the ESKAPE pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a harmful nosocomial pathogen associated with cystic fibrosis and burn wounds, encodes for a large number of LysR-type transcriptional regulator proteins. To understand how and why LTTR proteins evolved with such frequency and to establish whether any relationships exist within the distribution we set out to identify the patterns underpinning LTTR distribution in P. aeruginosa and to uncover cluster-based relationships within the pangenome. Comparative genomic studies revealed that in the JGI IMG database alone ~86 000 LTTRs are present across the sequenced genomes (n=699). They are widely distributed across the species, with core LTTRs present in >93 % of the genomes and accessory LTTRs present in <7 %. Analysis showed that subsets of core LTTRs can be classified as either variable (typically specific to P. aeruginosa) or conserved (and found to be distributed in other Pseudomonas species). Extending the analysis to the more extensive Pseudomonas database, PA14 rooted analysis confirmed the diversification patterns and revealed PqsR, the receptor for the Pseudomonas quinolone signal (PQS) and 2-heptyl-4-quinolone (HHQ) quorum-sensing signals, to be amongst the most variable in the dataset. Successful complementation of the PAO1 pqsR - mutant using representative variant pqsR sequences suggests a degree of structural promiscuity within the most variable of LTTRs, several of which play a prominent role in signalling and communication. These findings provide a new insight into the diversification of LTTR proteins within the P. aeruginosa species and suggests a functional significance to the cluster, conservation and distribution patterns identified.

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

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