Transcriptional Downregulation Research Articles

Abstract We139: 3D Chromatin Architectures and Transcription Regulation in Diabetic Endothelial Dysfunction

Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC high-glu ). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC high-glu and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC high-glu and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC high-glu . We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC high-glu significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.

Downregulation of lox transcription through Ang-1/Tie-2/STAT3 pathway inhibits endothelial cell injury in myocardial infarction mice

Downregulation of lox transcription through Ang-1/Tie-2/STAT3 pathway inhibits endothelial cell injury in myocardial infarction mice

A Phenotypic Approach to the Discovery of Potent G-Quadruplex Targeted Drugs.

G-quadruplex (G4) sequences, which can fold into higher-order G4 structures, are abundant in the human genome and are over-represented in the promoter regions of many genes involved in human cancer initiation, progression, and metastasis. They are plausible targets for G4-binding small molecules, which would, in the case of promoter G4s, result in the transcriptional downregulation of these genes. However, structural information is currently available on only a very small number of G4s and their ligand complexes. This limitation, coupled with the currently restricted information on the G4-containing genes involved in most complex human cancers, has led to the development of a phenotypic-led approach to G4 ligand drug discovery. This approach was illustrated by the discovery of several generations of tri- and tetra-substituted naphthalene diimide (ND) ligands that were found to show potent growth inhibition in pancreatic cancer cell lines and are active in in vivo models for this hard-to-treat disease. The cycles of discovery have culminated in a highly potent tetra-substituted ND derivative, QN-302, which is currently being evaluated in a Phase 1 clinical trial. The major genes whose expression has been down-regulated by QN-302 are presented here: all contain G4 propensity and have been found to be up-regulated in human pancreatic cancer. Some of these genes are also upregulated in other human cancers, supporting the hypothesis that QN-302 is a pan-G4 drug of potential utility beyond pancreatic cancer.

The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

Hyperuricemic nephropathy (HN) is a global metabolic disorder characterized by uric acid (UA) metabolism dysfunction, resulting in hyperuricemia (HUA) and tubulointerstitial fibrosis (TIF). Sodium-dependent glucose transporter 2 inhibitor, dapagliflozin, has shown potential in reducing serum UA levels in patients with chronic kidney disease (CKD), though its protective effects against HN remain uncertain. This study investigates the functional, pathological, and molecular changes in HN through histological, biochemical, and transcriptomic analyses in patients, HN mice, and UA-stimulated HK-2 cells. Findings indicate UA-induced tubular dysfunction and fibrotic activation, which dapagliflozin significantly mitigates. Transcriptomic analysis identifies estrogen-related receptor α (ERRα), a downregulated transcription factor in HN. ERRα knockin mice and ERRα-overexpressed HK-2 cells demonstrate UA resistance, while ERRα inhibition exacerbates UA effects. Dapagliflozin targets ERRα, activating the ERRα-organic anion transporter 1 (OAT1) axis to enhance UA excretion and reduce TIF. Furthermore, dapagliflozin ameliorates renal fibrosis in non-HN CKD models, underscoring the therapeutic significance of the ERRα-OAT1 axis in HN and CKD.

N6-methyladenosine writer METTL16-mediated alternative splicing and translation control are essential for murine spermatogenesis

BackgroundThe mitosis-to-meiosis switch during spermatogenesis requires dynamic changes in gene expression. However, the regulation of meiotic transcriptional and post-transcriptional machinery during this transition remains elusive.ResultsWe report that methyltransferase-like protein 16 (METTL16), an N6-methyladenosine (m6A) writer, is required for mitosis-to-meiosis transition during spermatogenesis. Germline conditional knockout of Mettl16 in male mice impairs spermatogonial differentiation and meiosis initiation. Mechanistically, METTL16 interacts with splicing factors to regulate the alternative splicing of meiosis-related genes such as Stag3. Ribosome profiling reveals that the translation efficiency of many meiotic genes is dysregulated in METTL16-deficient testes. m6A-sequencing shows that ablation of METTL16 causes upregulation of the m6A-enriched transcripts and downregulation of the m6A-depleted transcripts, similar to Meioc and/or Ythdc2 mutants. Further in vivo and in vitro experiments demonstrate that the methyltransferase activity site (PP185-186AA) of METTL16 is necessary for spermatogenesis.ConclusionsOur findings support a molecular model wherein the m6A writer METTL16-mediated alternative splicing and translation efficiency regulation are required to control the mitosis-to-meiosis germ cell fate decision in mice, with implications for understanding meiosis-related male fertility disorders.

Temporal changes in mouse hippocampus transcriptome after pilocarpine-induced seizures.

Status epilepticus (SE) is a seizure lasting more than 5 min that can have lethal consequences or lead to various neurological disorders, including epilepsy. Using a pilocarpine-induced SE model in mice we investigated temporal changes in the hippocampal transcriptome. We performed mRNA-seq and microRNA-seq analyses at various times after drug treatment. At 1 h after the start of seizures, hippocampal cells upregulated transcription of immediate early genes and genes involved in the IGF-1, ERK/MAPK and RNA-PolII/transcription pathways. At 8 h, we observed changes in the expression of genes associated with oxidative stress, overall transcription downregulation, particularly for genes related to mitochondrial structure and function, initiation of a stress response through regulation of ribosome and translation/EIF2 signaling, and upregulation of an inflammatory response. During the middle of the latent period, 36 h, we identified upregulation of membrane components, cholesterol synthesis enzymes, channels, and extracellular matrix (ECM), as well as an increased inflammatory response. At the end of the latent period, 120 h, most changes in expression were in genes involved in ion transport, membrane channels, and synapses. Notably, we also elucidated the involvement of novel pathways, such as cholesterol biosynthesis pathways, iron/BMP/ferroptosis pathways, and circadian rhythms signaling in SE and epileptogenesis. These temporal changes in metabolic reactions indicate an immediate response to injury followed by recovery and regeneration. CREB was identified as the main upstream regulator. Overall, our data provide new insights into molecular functions and cellular processes involved at different stages of seizures and offer potential avenues for effective therapeutic strategies.

P-789 Trophoblast organoids as a 3D model for the study of oxygen concentration during in vitro preimplantation embryo culture

Abstract Study question To determine if trophoblast organoids could represent a relevant robust model to study the impact of oxygen concentration on human preimplantation embryo development. Summary answer Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs differentially expressed. What is known already Our team has previously demonstrated that a biphasic (5-2%) O2 concentration strategy during preimplantation embryo culture (from Day 0 to Day 6) significantly improved blastulation and live birth rates in in vitro fertilization (IVF). We also identified differentially expressed RNAs between monophasic and biphasic O2 strategies through whole transcriptome analysis on human embryos donated for research. However, the number of embryos donated to research is limited, they are all genetically different, and their use iethically controversial, representing a significative drawback to experimentation. Latest studies on trophoblast organoids indicate that they could represent a relevant model for studying preimplantation embryo development. Study design, size, duration A prospective monocentric study approved by the Institutional Review Board (I.R.B.) was conducted between February 2022 and September 2023: four early first-trimester human placentas were collected after surgical abortions and used for derivation of organoids in a 3D culture system. The protocol for organoid culture is described in the study of Sheridan and al, 2020. Organoids were cultured with either a monophasic or a biphasic oxygen concentration strategy. Participants/materials, setting, methods Trophoblast organoids were either exposed to 5% oxygen concentration during 5 days or to 5% O2 for 3 days and to 2% O2 for 2 days. Total RNA extraction of 21 organoids for each condition was performed. After whole transcriptome analysis using human Clariom D arrays (ThermoFisher Scientific®), microarray data were normalized and differentially expressed RNAs were identified using the Transcriptome Analysis Console software; the genetic network was generated with Ingenuity Pathway Analysis (Qiagen®). Main results and the role of chance Transcriptomic analysis of trophoblast organoids cultured in monophasic (5%) or biphasic (5-2%) O2 concentration identified 1,184 RNAs that were differentially expressed depending on the O2 strategy (fold change >2 or <-2, p < 0.05), with most of them (75.1%, n = 889/1,184) being downregulated in the biphasic (5-2%) O2 group. IPA analysis highlighted downregulation of transcripts involved in cell survival and differentiation as well as upregulation of transcripts involved in embryo angiogenesis and vascularization of extraembryonic tissue. The transcript HIF1a was upregulated in organoids cultured in biphasic strategy compared to monophasic strategy (fold change 2, p = 0.0129). Comparison between differentially expressed transcripts in monophasic and biphasic strategies highlighted 22 transcripts in common between organoids and embryos donated to research from our previous study. Limitations, reasons for caution Our study was limited by a low sample size. Wider implications of the findings This innovative model of trophoblast organoids could help us to overcome the rarity as well as the genetic/epigenetic heterogeneity associated with IVF human embryos donated to the research, thus leading to the realization of large-scale experimentations and promoting the identification of new non-invasive predictive biomarkers for IVF. Trial registration number not applicable

Downregulation of a Phi class glutathione S-transferase gene in transgenic torenia yielded pale flower color.

The members of glutathione S-transferase (GST) belonging to the Phi class of the GST family are known to play a role in anthocyanin transport to the vacuole. We isolated a GST orthologue from the torenia petal cDNA library. Transgenic plants transcribing GST double stranded RNA were generated from a torenia cultivar having blue flowers. These plants exhibited a range of flower colors, from blue to almost white. Quantitative RT-PCR confirmed the downregulation of the GST transcript, accompanied by a decrease in anthocyanin levels in the petals of the transgenic plants, whereas flavone levels remained unchanged. These results suggest that GST is involved in anthocyanin transport in torenia petals, and that anthocyanins and flavones are likely transported to the vacuole through different mechanisms.

Phosphorus limitation intensifies heat-stress effects on the potential mutualistic capacity in the coral-derived Symbiodinium

Coral reef ecosystems have been severely ravaged by global warming and eutrophication. Eutrophication often originates from nitrogen (N) overloading that creates stoichiometric phosphorus (P) limitation, which can be aggravated by sea surface temperature rises that enhances stratification. However, how P-limitation interacts with thermal stress to impact coral-Symbiodiniaceae mutualism is poorly understood and underexplored. Here, we investigated the effect of P-limitation (P-depleted vs. P-replete) superimposed on heat stress (31 °C vs. 25 °C) on a Symbiodinium strain newly isolated from the coral host by a 14-day incubation experiment. The heat and P-limitation co-stress induced an increase in alkaline phosphatase activity and reppressed cell division, photosynthetic efficiency, and expression of N uptake and assimilation genes. Moreover, P limitation intensified downregulation of carbon fixation (light and dark reaction) and metabolism (glycolysis) pathways in heat stressed Symbiodinium. Notably, co-stress elicited a marked transcriptional downregulation of genes encoding photosynthates transporters and microbe-associated molecular patterns, potentially undermining the mutualism potential. This work sheds light on the interactive effects of P-limitation and heat stress on coral symbionts, indicating that nutrient imbalance in the coral reef ecosystem can intensify heat-stress effects on the mutualistic capacity of Symbiodiniaceae.

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the “simultaneous inhibition of a paralog pair” concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

Dynamic network biomarker of treg cells differentiation critical state with neoadjuvant treatment in LUSC.

e20063 Background: Regulatory T cells (Tregs) suppress immune responses and can promote tumor growth and metastasis during cancer progression. Studies have shown that tumor-infiltrating Tregs upregulate inhibitory gene programs after immune checkpoint blockade therapy. Targeting Tregs is believed to be a valuable approach to enhance the effectiveness of immunotherapy. However, the precise role of Tregs in the tumor microenvironment, their connection to treatment resistance mechanisms, and how changes in their immune activity impact tumor treatment are still not well understood. Methods: Single-cell data and clinical information were obtained from the Gene Expression Omnibus database with the accession number GSE176021 for 4 patients diagnosed with lung squamous cell carcinoma (LUSC) who received neoadjuvant anti-PD-1 therapy, including 2 patients with major pathologic response (MPR) and 2 patients with non-MPR. BioTIP was employed to identify critical transition signals of the Treg cell differentiation trajectory constructed by Monocle2. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set variation analysis were performed to understand biological processes and pathways. Additionally, potential transcription factors were identified using Single-Cell rEgulatory Network Inference and Clustering, and CellChat analysis was conducted to evaluate possible interactions among cell populations. Results: The study constructed a pseudo-temporal differentiation trajectory of Tregs in LUSC patients, and Treg subpopulations during the critical transition period were identified based on the dynamic network biomarker method. Notably, subpopulations of Treg cells clustering MPR or non-MPR samples were located at different ends of the differentiation trajectory, showing variations in Treg-related pathways and transcription factor regulation. The critical transition subpopulation exhibited high expression of glycolysis, TCA cycle, and natural killer cell receptors, along with upregulation of the CEBPB transcription factor activity and downregulation of ARNTL activity. SELPLG-SELL, CD99-CD99, and MHC-II-CD4 were identified as potential key signals that may affect therapeutic effect, primarily influencing cell adhesion, the immune suppressive activity of Treg cells, and T cell response. Conclusions: In conclusion, this study utilized single-cell data analysis to uncover the dynamic regulation of Treg cells in LUSC, identifying critical transition Treg subpopulations. Notably, our findings suggest the involvement of the transcription factors CEBPB and ARNTL in regulating Treg activity. These novel insights shed light on potential strategies for targeted therapies and provide valuable information regarding resistance mechanisms associated with Treg cells.

DNA Damage Checkpoints Govern Global Gene Transcription and Exhibit Species-Specific Regulation on HOF1 in Candida albicans.

DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in Saccharomyces cerevisiae and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen Candida albicans, their targets remain poorly understood. In this study, we explored the impact of deleting RAD53 on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of RAD53 only affected a limited number of known DNA damage-responsive genes, including MRV6 and HMX1. Unlike S. cerevisiae, the downregulation of HOF1 transcription in C. albicans under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of HOF1 transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of RAD53. Furthermore, Rad53 was observed to directly interact with the promoter region of HOF1, thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in C. albicans and show species-specific regulation on HOF1; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.

Identification of RNAi-Related Genes and Transcriptome Assembly of Loblolly Pine (Pinus taeda, L.) Seedlings Exposed to Insect-Specific dsRNA

Considerable research has focused on gene silencing in tree-feeding insects, but how trees recognize and process double-stranded RNA (dsRNA) engineered to target plant pests is unknown. We performed transcriptomic assembly, preliminary differential expression analysis, and in silico annotation on loblolly pine (Pinus taeda, L.) seedlings exposed to southern pine beetle-specific dsRNA. This pilot study sought to elucidate the baseline response of seedlings challenged with insect-specific dsRNA. Treated and untreated seedlings were sequenced and following transcriptome assembly 20 RNAi-related proteins (RRPs) were annotated. Differential gene expression analysis conducted using DESeq2 followed by pathway enrichment revealed 7131 differentially expressed transcripts, of which 33% were upregulated and 67% were downregulated. Only two RRPs selected for analysis were upregulated in treated seedlings, showing a lack of detectable RNAi response with our methodology. Beyond RNAi-related proteins, pathway enrichment mapped to immune response systems and genetic and cellular processing. Upregulated transcripts included autophagy, amino sugar and nucleotide sugar metabolism, and plant hormone signal transduction. Downregulated transcripts included RNA degradation and fatty acid metabolism pathways. Multiple DICER-LIKE and ARGONAUTE proteins were also annotated in five other North American pines, revealing diversity among these crucial proteins. Understanding host plant response to RNAi-mediated pest control is essential to further develop this technology against tree pests.

Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L

BackgroundCerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) – a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer.MethodsOvarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins.ResultsFor each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level.ConclusionsOur study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

Combining WO3@AuNPs with Poly(amidoamine) Allows Sensitive Electrochemical Detection of DR1 Based on Dual Signal Amplification.

Down-regulator of transcription 1 (DR1) is considered as a biomarker of hashimoto's thyroiditis (HT), which is a risk factor for thyroid cancer. Here, a label-free electrochemical biosensor for DR1 detection was constructed based on polyamidoamine (PAMAM) polymer and the nanocomposite (WO3@AuNPs) composed of tungsten trioxide (WO3) and gold nanoparticles (AuNPs). WO3@AuNPs was obtained by combining monolayer WO3 nanosheets, which has high conductivity, and AuNPs. The modification of WO3@AuNPs can not only increase the conductivity of the electrode but also provide more active sites for signaling units, thus greatly improve the sensitivity of the sensor. The polymer PAMAM is biocompatible and non-immunogenic, and its end functional group can bind to the target molecules, providing them with more binding sites and thus improving the sensitivity of the sensor. Under optimal conditions, the label-free biosensor showed a good linear relationship between the logarithm of DR1 concentration and the impedance in the range of 10 fg ⋅ mL-1 to 100 ng ⋅ mL-1, with a detection limit as low as 0.3 fg ⋅ mL-1. Besides, this label-free electrochemical platform exhibited satisfactory selectivity and anti-interference capability in human serum samples. Therefore, this method has considerable potential in clinical detection of DR1.

Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress

Mechanisms of functional cross-talk between global transcriptional repression and efficient DNA damage repair during genotoxic stress are poorly known. In this study, using human AF9 as representative of Super Elongation Complex (SEC) components, we delineate detailed mechanisms of these processes. Mechanistically, we describe that Poly-Serine domain-mediated oligomerization is pre-requisite for AF9 YEATS domain-mediated TFIID interaction-dependent SEC recruitment at the promoter-proximal region for release of paused RNA polymerase II. Interestingly, during genotoxic stress, CaMKII-mediated phosphorylation-dependent nuclear export of AF9-specific deacetylase HDAC5 enhances concomitant PCAF-mediated acetylation of K339 residue. This causes monomerization of AF9 and reduces TFIID interaction for transcriptional downregulation. Furthermore, the K339 acetylation-dependent enhanced AF9-DNA-PKc interaction leads to phosphorylation at S395 residue which reduces AF9-SEC interaction resulting in transcriptional downregulation and efficient repair of DNA damage. After repair, nuclear re-entry of HDAC5 reduces AF9 acetylation and restores its TFIID and SEC interaction to restart transcription.

N-acetylcysteine alleviates arsenic trioxide-induced reductions in hepatic catalase gene expression both in vitro and in vivo

Arsenic trioxide (ATO), one of the oldest and most frequently used poisons, is well-known in forensic science for inducing hepatotoxicity. The regulation of peroxisomal antioxidative enzyme catalase (CAT) involves intricate mechanisms at both transcriptional and post-transcriptional levels. However, the molecular mechanisms underlying the regulation of CAT gene expression in hepatic cells remain elusive. Furthermore, the regulation of CAT gene expression evident in animals administered with ATO in vivo is not well-explored, although several studies have revealed ATO-induced reductions in CAT enzymatic activity in rat livers. In this study, we revealed ATO-dependent reductions in CAT gene expression in both rat liver and Huh-7 human hepatoma cells. Our results indicate that the decline in CAT enzymatic activity can be attributed, at least in part, to the downregulation of its gene expression. The ATO-induced reduction in CAT expression was concurrent with the reduction in peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator (PGC)-1α and inactivation of PPARγ, both considered as positive regulators of CAT gene expression. Moreover, antioxidant N-acetylcysteine (NAC) demonstrated the capability to alleviate the downregulation of CAT gene expression both in vivo and in vitro. Additionally, NAC played a role in alleviating ATO-induced hepatotoxicity, potentially by mitigating the transcriptional downregulation of the CAT gene. Altogether, these results indicate that ATO exerts toxicity by inhibiting the antioxidant defense mechanism, which may be useful for forensic diagnosis of arsenic poisoning and clinical treatment of mitigating ATO-induced hepatotoxicity.

Tribbles pseudokinase 3 promoted renal fibrosis by regulating the expression of DNA damage-inducible transcript 3 in diabetic nephropathy.

Diabetic nephropathy (DN) is a severe complication of prolonged diabetes, impacting millions worldwide with an increasing incidence. This study investigates the role of tribbles pseudokinase 3 (TRIB3), a protein implicated in the progression of DN, focusing on its mechanisms underlying glomerular damage. Through analysis of the Gene Expression Omnibus (GEO) database, we identified TRIB, among differentially expressed genes (DEGs) in streptozotocin (STZ)-treated C57BL/6J mice. Both in vitro and in vivo experiments were conducted to examine the effects of TRIB3 inhibition on high glucose (HG)-induced damage in podocytes and DN mouse models. The results demonstrated that TRIB3 inhibition reduced inflammatory responses and extracellular matrix (ECM) production inMPC5 cells, mediated by the downregulation of DNA damage-inducible transcript 3 (DDIT3) - a critical regulator of proinflammatory cytokine secretion and ECM synthesis. Inhibiting TRIB3 decreased inflammatory factors and ECM deposition in diabetic mice in vivo, confirming its pivotal role in DN pathogenesis. These findings indicate that TRIB3 and its interaction with DDIT3 contribute significantly to DN by promoting inflammatory cascades and ECM accumulation, presenting potential therapeutic targets for managing the disease.

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells.

Defects in the FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone composed of SSRP1 and SUPT16H, are implicated in intellectual disability. Here, we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain. We show that the FACT complex binds to the promoter region of the estrogen-related receptor (ERR) gene and positively regulates ERR expression. ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis. Dysfunction of the FACT complex leads to the downregulation of ERR transcription, resulting in a decreased ratio of glycolysis to oxidative phosphorylation (G/O) in neuroblasts. Consequently, neuroblasts exhibit smaller cell sizes, lower proliferation potential, and altered cell fates. Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex. Thus, the G/O ratio, mediated by the FACT complex, plays a crucial role in neuroblast cell fate maintenance. Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.