Gene Transcriptional Activity Research Articles

CpSmt3, an ortholog of small ubiquitin-like modifier, is essential for growth, organelle function, virulence, and antiviral defense in Cryphonectria parasitica.

SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored. A comprehensive analysis composed of proteomics, affinity pull-down, molecular and cellular approaches was performed to explore the roles of SUMOylation in Cryphonectria parasitica, the fungal pathogen responsible for chestnut blight. CpSmt3, the gene encoding the SUMO protein CpSmt3 in C. parasitica was identified and characterized. Deletion of the CpSmt3 gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The ΔCpSmt3 deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of CpSmt3 reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of CpRho1 resulted in a phenotype similar to that of ΔCpSmt3, while overexpression of CpRho1 could partly rescue some of the prominent defects in ΔCpSmt3. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in C. parasitica.

MYB4 is the best candidate transcription factor involved in pinosylvin stilbene biosynthesis in Pinus strobus L. cells by fungal elicitor treatment

Key messageDihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME) are pinosylvin derivatives that show high nematicidal activity against pine wood nematodes (PWNs). Here, we found that fungal elicitor treatment boosted the production of DPME and PME in cultured Pinus strobus L. cells and investigated the transcription factors (TFs) regulating the genes in the pinosylvin stilbenoid biosynthesis pathway.ContextThe discovery of TFs involved in the synthesis of DPME and PME provides an important clue to understanding the pinosylvin stilbenoid synthesis in pine plants.AimsWe investigated the best fungal elicitor for the production of DPME and PME and the transcriptional activities of genes involved in PME and DPME biosynthesis in P. strobus L. cells after fungal elicitor treatment.MethodsThe content of DPME and PME in P. strobus cells was examined after treatment with fungal elicitors prepared from seven different species of fungi. Moreover, the role of fungal elicitors in the transcriptional activity of genes involved in DPME and PME biosynthesis was investigated by transcriptome analysis using RNA sequencing.ResultsPenicillium chrysogenum Thorn was the most efficient fungal elicitor for the production of DPME and PME in P. strobus cells among the other fungal species. The accumulation of DPME and PME in P. strobus cells after P. chrysogenum elicitor treatment increased 12.7-fold and 23.7-fold, respectively, compared to the control. Transcriptome analysis revealed that fungal elicitor treatment resulted in enhanced transcription of the PAL, 4CL, STS, PMT, and ACC genes, which are involved in PME and DPME biosynthesis. Some transcription factors belonging to the bHLH, MYB, WRKY, and ERF families showed a high transcription rate after fungal elicitor treatment.ConclusionsWe found that the fungal elicitor is a strong inducer of the accumulation of pinosylvin derivatives in the cells of P. strobus. We selected one unigene (c133966_g3_i1 in the MYB family) as the best candidate TF that regulates the DPME and PME biosynthesis in P. strobus by transcriptome analysis.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Overexpression of ZNF169 promotes the growth and proliferation of colorectal cancer cells via the upregulation of ANKZF1.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The 5‑year survival rate of patients diagnosed with the early stages of the disease is markedly higher than that of patients in the advanced stages. Therefore, identifying novel biomarkers and drug targets for CRC is critical for clinical practice. Zinc finger protein 169 (ZNF169) is a crucial transcription factor, and its role in CRC remains to be explored. The present study aimed to investigate the clinical relevance, function and underlying mechanisms of ZNF169 in CRC growth and proliferation. The Cancer Genome Atlas (TCGA) database was utilized to analyze the clinical relevance of ZNF169 in patients with CRC. Immunohistochemical staining was performed on tissue samples from patients with CRC to detect the expression of ZNF169. The HCT‑116, HT‑29 and RKO cell lines were employed for in vitro experiments. The overexpression and knockdown of ZNF169 were achieved by transfecting the cells with lentivirus and small interfering RNAs, respectively. Cell Counting Kit‑8, colony formation and EdU staining assays were applied to investigate the function of ZNF169 in CRC cells. Dual luciferase activity and chromatin immunoprecipitation (ChIP)‑quantitative PCR (qPCR) assays were performed to identify the regulatory effects of ZNF169 on the ankyrin repeat and zinc‑finger domain‑containing 1 (ANKZF1; also known as ZNF744) gene. Reverse transcription‑quantitative PCR and western blot analysis were performed to measure mRNA and protein expression, respectively. The analysis of TCGA data revealed a positive correlation between ZNF169 and ANKZF1, with the overexpression of ANKZF1 being associated with a poor prognosis of patients with CRC. The experimental results demonstrated that ZNF169 was expression upregulated in CRC tissue compared with that in normal colon tissue. Gain‑of‑function and loss‑of‑function experiments revealed that ZNF169 enhanced the intensity of EdU staining, promoting the growth and proliferation of CRC cells. Furthermore, the overexpression of ZNF169 potentiated the transcriptional activity of the ANKZF1 gene, while the knockdown of ZNF169 produced the opposite results. ChIP‑qPCR confirmed the interaction between ZNF169 and the promoter sequence of ANKZF1. Rescue experiments revealed that ZNF169 accelerated CRC cell growth and proliferation through the upregulation of ANKZF1. Furthermore, there was a positive correlation identified between ZNF169 and ANKZF1, and upregulation of ANKZF1 expression was associated with the poor prognosis of patients with CRC. On the whole, the present study demonstrates that ZNF169 contributes to CRC malignancy by potentiating the expression of ANKZF1. Thus, the regulation of ZNF169 and/or ANKZF1 expression may represent a viable strategy for the treatment patients with CRC with a high expression of ZNF169.

Adaptive expansion of ERVK solo-LTRs is associated with Passeriformes speciation events

Endogenous retroviruses (ERVs) are ancient retroviral remnants integrated in host genomes, and commonly deleted through unequal homologous recombination, leaving solitary long terminal repeats (solo-LTRs). This study, analysing the genomes of 362 bird species and their reptilian and mammalian outgroups, reveals an unusually higher level of solo-LTRs formation in birds, indicating evolutionary forces might have purged ERVs during evolution. Strikingly in the order Passeriformes, and especially the parvorder Passerida, endogenous retrovirus K (ERVK) solo-LTRs showed bursts of formation and recurrent accumulations coinciding with speciation events over past 22 million years. Moreover, our results indicate that the ongoing expansion of ERVK solo-LTRs in these bird species, marked by high transcriptional activity of ERVK retroviral genes in reproductive organs, caused variation of solo-LTRs between individual zebra finches. We experimentally demonstrated that cis-regulatory activity of recently evolved ERVK solo-LTRs may significantly increase the expression level of ITGA2 in the brain of zebra finches compared to chickens. These findings suggest that ERVK solo-LTRs expansion may introduce novel genomic sequences acting as cis-regulatory elements and contribute to adaptive evolution. Overall, our results underscore that the residual sequences of ancient retroviruses could influence the adaptive diversification of species by regulating host gene expression.

GSK0660 enhances antitumor immunotherapy by reducing PD-L1 expression

Blockade of PD-1/PD-L1 immune checkpoint is wildly used for multiple types of cancer treatment, while the low response rate for patients is still completely unknown. As nuclear hormone receptor, PPARδ (peroxisome-proliferator-activated receptor) regulates cell proliferation, inflammation, and tumor progression, while the effect of PPARδ on tumor immune escape is still unclear. Here we found that PPARδ antagonist GSK0660 significantly reduced colon cancer cell PD-L1 protein and gene expression. Luciferase analysis showed that GSK0660 decreased PD-L1 gene transcription activity. Moreover, reduced PD-L1 expression in colon cancer cells led to increased T cell activity. Further analysis showed that GSK0660 decreased PD-L1 expression in a PPARδ dependent manner. Implanted tumor model analysis showed that GSK0660 inhibited tumor immune escape and the combined PD-1 antibody with GSK0660 effectively enhanced colorectal cancer immunotherapy. These findings suggest that GSK0660 treatment could be an effective strategy for cancer immunotherapy.

Abstract 963: Comprehensive epigenomic profiling from plasma to inform therapy selection: A proof-of-concept study in cancer

Abstract BACKGROUND: Analysis of genomic alterations in circulating tumor DNA is gaining traction in clinical oncology and can provide insights into tumor biology without the need for invasive tissue sampling. In addition to genetic alterations, epigenomic reprogramming leads to transcriptional dysregulation and drives cancer phenotypes including molecular subtypes, histologic subtypes, and mechanisms resistance. As current liquid biopsy assays fail to effectively capture transcriptional biology, new approaches are needed to examine the transcriptional status of therapeutic gene targets and pathways including those relevant to rapidly emerging classes such as antibody-drug conjugates (ADC) and radio-conjugates. METHODS: We developed a novel multimodal assay that reveals genome-wide transcriptional activity of promoters, enhancers from chromatin and surveys the DNA methylome using 1ml of patient plasma. We applied this assay to patient plasma samples from across 15 tumor types generating 7000 genome-wide transcriptional profiles. RESULTS: We confirmed a significant correlation between epigenomic signals of transcriptional activation and gene expression from RNA-seq (Promoter vs RNA-seq: ⍴ =0.95, p<0.05; DNA methylation vs RNA-seq: ⍴=-0.16, p<0.05). We identified transcriptional activation in cancer patient plasma of well-established cancer-associated biomarkers (e.g. MET, DLL3, PSMA) in a tumor-specific manner. Epigenomic profiling simultaneously inferred the expression of multiple pertinent ADC targets currently under investigation (MET, HER2, HER3, TROP2, B7H4 in breast cancer), and SLFN11, a known biomarker for sensitivity to these agents. Unlike targeted panels, the platform assesses transcriptional regulation genome-wide, enabling the assessment of transcriptional activity of select genes as well as related pathways. For example, a multimodal classifier robustly predicted HER2 status across metastatic breast cancer plasma samples based on features both within the HER2 amplicon as well as HER2-distal genes that are activated in HER2+ cancers (AUC = 0.93, 0.84-1.0 95% CI). The multimodal classifier out-performed individual analyte-based classifiers (AUCPromoter+Enhancer = 0.85, 0.72-0.98 95% CI; AUCDNA-methylation = 0.69, 0.48-0.90 95% CI). Epigenomic signatures were also used to delineate multiple mechanisms of resistance to androgen receptor signaling inhibition in prostate cancer, including reactivation of the androgen receptor, glucocorticoid signaling bypass, and histological transformation to a neuroendocrine subtype. CONCLUSION: This comprehensive epigenomic liquid biopsy assay provides a platform for simultaneous genome-wide transcriptional assessment of disease and therapy relevant genes and pathways, representing a paradigm shift in our ability to longitudinally monitor cancer in individual patients. Citation Format: Travis Clark, Ji-Heui Seo, Anthony D'Ippolito, Aparna Gorthi, Jonathan Beagan, Jamey Guess, Amy Donahue, Mandy Greene, Alyssa Lau, Fernando Ramirez, Kristian Cibulskis, Hyun-Hwan Jeong, Matthew Davidsohn, Gitanjali Lakshminarayanam, Michael Coyne, Baovy Tran, Tyrone Tamakloe, Hathairat Sawaengsri, Marc Eid, Karl Semaan, Brad Fortunato, Corrie A. Painter, Juliann Chmielecki, Toni K. Choueiri, J Carl Barrett, Geoff Otto, Matthew L. Freedman, Matthew L. Eaton, Sylvan C. Baca. Comprehensive epigenomic profiling from plasma to inform therapy selection: A proof-of-concept study in cancer [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 963.

Effect of chronic intake of cadmium chloride on the transcriptional activity of metallothionein and zinc transporter genes

Introduction. Cadmium chloride is an inorganic compound containing cadmium, a heavy metal that is one of the active environmental pollutants today. Damage to organs in experimental animals due to cadmium poisoning is similar to that in humans. In this work, the activity of metallothionein and zinc transporters genes was studied in a chronic model of cadmium-induced poisoning in experimental animals.
 Materials and methods. The experiment was carried out using seventy two individuals of white inbred rats of both sexes, the average weight of which was 215 g. Animals from four groups were injected with a solution of cadmium chloride in four different doses, respectively, individuals of the fifth group, the control group, received an equimolar volume of pure water. The objects of the study were the kidneys and livers of rats, removed after the animals were withdrawn from the experiment. Next, the activity of the Mt1A, Mt2A, Mt3A, Zip1 and Znt1 genes was analyzed in organ samples using real-time PCR.
 Results. Significant increases in the expression multiplicity of Mt1A, Mt2A and Mt3A metallothionein genes in the kidneys at different doses of the toxicant were revealed. In liver samples, a decrease in the expression of the Mt2A gene was found in the experimental group exposed to cadmium chloride at a dose 
 of 0.1 mg/kg (p<0.05). For the Znt1 gene in rat liver tissue, there was a statistically significant decrease in expression at a dose of 0.001 mg/kg (p<0.05) 
 and, conversely, an increase at doses of 0.1 and 1 mg/kg (p<0.05) compared to the control group. Analysis of the level of transcripts of the Zip1 gene in the kidneys and liver after 6 months of inoculation with the toxicant in the presented doses did not reveal statistically significant differences between the groups.
 Limitations. Laboratory animals of the only biological species were used for the experiment. Four doses of the cadmium salt alone were evaluated.
 Conclusion. The results obtained allow concluding that the level of expression of the Mt1A, Mt2A and Mt3A genes in the kidneys can play the role of a diagnostic marker in chronic poisoning with the toxicant under study.

An Imbalance in Histone Modifiers Induces tRNA-Cys-GCA Overexpression and tRF-27 Accumulation by Attenuating Promoter H3K27me3 in Primary Trastuzumab-Resistant Breast Cancer.

tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments.

The influence of mitochondria-targeted antioxidants, plastoquinone derivatives on the resistance of grain crops to water deficiency

The article presents the results of a study of the influence of plastoquinone derivatives: 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) and 10-(6′-methylplastoquinonyl) decyltriphenylphosphonium (SkQ3) on the resistance of grain crops to soil moisture deficiency. The object of the study were seedlings of spring barley (Hordeum vulgare L.) cultivar “Sokol” and winter wheat (Triticum aestivum L.) cultivar “Kalym”. The growth rates of plants were studied for 14 days under normal conditions and with a lack of moisture, pre-treated with 2.5 nm solutions of SkQ1 and SkQ3. It has been shown that treating seeds with solutions of SkQ1 and SkQ3 increases the growth rate of the studied crops under conditions of lack of moisture compared to the control. Also, in the roots and leaves of 14-day-old barley seedlings, an analysis of the transcriptional activity of the genes SodA1, SodB, GR, GST1, GST6, Cat1, Cat2, Apx1, encoding oxidative stress proteins, was carried out. It was shown that a decrease in soil moisture by 1.8 and 2.0 times compared to the control reduces to varying degrees the weight of roots and leaves in 14-day-old barley seedlings and increases the transcriptional activity of all studied genes in both leaves and roots. Treatment of seeds with solutions of SkQ1 and SkQ3 under conditions of moisture deficiency increases the dry weight of barley roots and leaves and reduces the transcriptional activity of the studied genes in the roots. Keywords: PLASTOQUINONE DERIVATIVES, SkQ1, SkQ3, MITOCHONDRIA-TARGETED ANTIOXIDANTS, ROS, SOIL MOISTURE DEFICIENCY, STABILITY, TRANSCRIPTIONAL GENE ACTIVITY

The landscape of transcription factor promoter activity during vegetative development in Marchantia.

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterizing the transcriptional activity of TF genes in space and time is a critical step toward understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analyzed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem-cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics.

CENPA promotes glutamine metabolism and tumor progression by up-regulating SLC38A1 in endometrial cancer

Glutamine addiction is a significant hallmark of metabolic reprogramming in tumors and is crucial to the progression of cancer. Nevertheless, the regulatory mechanisms of glutamine metabolism in endometrial cancer (EC) remains elusive. In this research, we found that elevated expression of CENPA and solute carrier family 38 member 1 (SLC38A1) were firmly associated with worse clinical stage and unfavorable outcomes in EC patients. In addition, ectopic overexpression or silencing of CENPA could either enhance or diminish glutamine metabolism and tumor progression in EC. Mechanistically, CENPA directly regulated the transcriptional activity of the target gene, SLC38A1, leading to enhanced glutamine uptake and metabolism, thereby promoting EC progression. Notably, a prognostic model utilizing the expression levels of CENPA and SLC38A1 genes independently emerged as a prognostic factor for EC. More importantly, CENPA and SLC38A1 were significantly elevated and positively correlated, as well as indicative of poor prognosis in multiple cancers. In brief, our study confirmed that CENPA is a critical transcription factor involved in glutamine metabolism and tumor progression through modulating SLC38A1. This revelation suggests that targeting CENPA could be an appealing therapeutic approach to address pan-cancer glutamine addiction.

Investigating the Role of OrbF in Biofilm Biosynthesis and Regulation of Biofilm-Associated Genes in Bacillus cereus BC1.

Bacillus cereus (B. cereus), a prevalent foodborne pathogen, constitutes a substantial risk to food safety due to its pronounced resilience under adverse environmental conditions such as elevated temperatures and ultraviolet radiation. This resilience can be attributed to its capacity for biofilm synthesis and sustained high viability. Our research aimed to elucidate the mechanisms governing biofilm biosynthesis in B. cereus. To this end, we constructed a 5088-mutant library of the B. cereus strain BC1 utilizing the transposon TnYLB-1. Systematic screening of this library yielded mutants exhibiting diminished biofilm formation capabilities. Twenty-four genes associated with the biofilm synthesis were identified by reverse PCR in these mutants, notably revealing a significant reduction in biofilm synthesis upon disruption of the orbF gene in B. cereus BC1. Comparative analysis between the wild type and orbF-deficient BC1 strains (BC1ΔorbF) indicated a marked downregulation (decreased by 11.7% to 96.7%) in the expression of genes implicated in biofilm formation, flagellar assembly, and bacterial chemotaxis in the BC1ΔorbF. Electrophoretic mobility shift assay (EMSA) further corroborated the role of OrbF, demonstrating its binding to the promoter region of the biofilm gene cluster, subsequently leading to the suppression of transcriptional activity of biofilm-associated genes in B. cereus BC1. Our findings underscore the pivotal role of orbF in biofilm biosynthesis in B. cereus, highlighting its potential as a target for strategies aimed at mitigating biofilm formation in this pathogen.

A26 BIOLOGICAL CHARACTERIZATION OF THE INTERACTION BETWEEN SIRT1 AND HNF4Α2 IN INTESTINAL EPITHELIAL CELLS

Abstract Background Colorectal cancer (CRC) is the third most common cancer in Canada. HNF4A locus is amplified in CRC, while additional reports suggest that hepatocyte nuclear factor 4 alpha (HNF4α) is associated with increased proliferation and disease development. This dual role as a tumor suppressor or oncoprotein might be due to the production of 12 spliced isoforms with structural differences and variable tissue expression. It suggests distinct functions for each isoform according to their specific interaction complexes. However, little is known about the nature of these protein complexes and their biological functions during intestinal physiopathology. Recently, using a BioID2 and mass spectrometry approach, our laboratory showed a possible interaction between HNF4α2 and sirtuin 1 (SIRT1) in intestinal epithelial cells from a colon carcinoma (HCT116). HNF4α2 is one of the most potent isoforms in the control of transcriptional activity of multiple intestinal epithelial genes related to regulating cell death, angiogenesis, apoptosis, response to injury, and response to drugs, among others. Aims To characterize the possible interaction between HNF4α2 and SIRT1 in intestinal epithelial cells at the biological level. Methods We performed SIRT1 knockdown using shRNAs in HCT116 cells expressing the HNF4α2 isoform in an inducible manner by doxycycline (DOX). Subsequently, we conducted a proximity ligation assay to validate the interaction between HNF4α2 and SIRT1 in HCT116 cells. Additionally, protein purification was performed to validate their physical interaction through EMSA. A transcriptome analysis was carried out using RNA-seq to define the biological processes involved in the interaction between the two proteins. Results We observed an interaction signal between HNF4α2 and SIRT1 in HCT116 cells, similar to that observed for IRFBB2, whose interaction with HNF4α2 has been previously validated. This signal decreased when cells were treated with shRNA for SIRT1 and was not observed in cells not induced by DOX. EMSA analyses revealed a direct interaction signal between HNF4α2 and SIRT1. RNA-seq analyses showed a loss in the regulation of over 95% of genes targeted by HNF4α2 when cells were treated with shRNA for SIRT1. An enrichment analysis for GO annotations using ShyniGO v. 0.77 software showed an enrichment of genes for biological processes such as apoptosis, inflammation, cell migration, cell invasion, cell death, and various cancer-related signaling pathways. Conclusions SIRT1 displays physical interaction with HNF4α2 and significantly affects the transcriptional regulation of this isoform in the context of epithelial cells. Their interaction is involved in processes related to intestinal inflammation and cancer progression. Pharmacological targeting of SIRT1 could represent a viable strategy in treating CRC and other intestinal diseases Funding Agencies CIHRNSERC

Novel FOXL2 variants in two Chinese families with blepharophimosis, ptosis, and epicanthus inversus syndrome.

Introduction: Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare inherited disorder. This study was aimed to identify and functionally validate FOXL2 variants in two Chinese families with BPES. Methods: The proband and his family members were subjected to whole-exome sequencing to identify disease-associated variants. Several bioinformatic tools were used to computationally predict altered proteins. In vitro functional assays were conducted by transfecting wild-type and mutant FOXL2 cDNAs into HEK-293 cells, followed by subcellular localization assays, luciferase reporter gene assays, and quantitative real-time polymerase chain reaction. Results: The clinical features of BPES, including small palpebral fissures, ptosis, telecanthus, and epicanthus inversus, were present in all affected patients. Two novel mutations were detected, c.292T>A and c.383G>T. Whole-exome sequencing analysis and prediction software suggested that these mutations were pathogenic. Functional studies showed that these two point mutations decreased FOXL2 protein expression, resulting in subcellular mislocalization and aberrant transcriptional activity of the steroidogenic acute regulatory protein gene promoter. Conclusion: Our results add to the current understanding of known FOXL2 variants in, and our in vitro experiments provide reference data and insights into the etiology of BPES. Further studies are needed to identify the possible mechanisms underlying the action of this mutation on the development of BPES.

PMP2 regulates myelin thickening and ATP production during remyelination.

It is well established that axonal Neuregulin 1 type 3 (NRG1t3) regulates developmental myelin formation as well as EGR2-dependent gene activation and lipid synthesis. However, in peripheral neuropathy disease context, elevated axonal NRG1t3 improves remyelination and myelin sheath thickness without increasing Egr2 expression or activity, and without affecting the transcriptional activity of canonical myelination genes. Surprisingly, Pmp2, encoding for a myelin fatty acid binding protein, is the only gene whose expression increases in Schwann cells following overexpression of axonal NRG1t3. Here, we demonstrate PMP2 expression is directly regulated by NRG1t3 active form, following proteolytic cleavage. Then, using a transgenic mouse model overexpressing axonal NRG1t3 (NRG1t3OE) and knocked out for PMP2, we demonstrate that PMP2 is required for NRG1t3-mediated remyelination. We demonstrate that the sustained expression of Pmp2 in NRG1t3OE mice enhances the fatty acid uptake in sciatic nerve fibers and the mitochondrial ATP production in Schwann cells. In sum, our findings demonstrate that PMP2 is a direct downstream mediator of NRG1t3 and that the modulation of PMP2 downstream NRG1t3 activation has distinct effects on Schwann cell function during developmental myelination and remyelination.

Lipidomics, transcription analysis, and hormone profiling unveil the role of CsLOX6 in MeJA biosynthesis during black tea processing.

Jasmonates, such as jasmonic acid (JA) and methyl jasmonate (MeJA), are crucial aspect of black tea quality. However, lipids species, hormones, and genes regulated mechanism in the jasmonate biosynthesis during black tea processing are lacking. In this study, we employed lipidomics, hormone metabolism analysis, and transcriptome profiling of genes associated with the MeJA biosynthesis pathway to investigate these factors. The contents of lipids GLs, PLs, and TAG are decreased, accompanied by the main lipids species reduced during black tea processing. Galactolipids, primarily 34:3/36:6/36:3 DGDG and 36:6/36:5/36:4 MGDG, are transformed into massive MeJA and JA in black tea processing, accompanied by the decreased SA, MeSA, IAA, and BA and increased zeatin. Additionally, the transcriptional activity of the primary genes in MeJA biosynthesis pathway exhibited downregulated trends except for AOS and OPR and non-primary genes tend to be a little high or have fluctuation of expression. Coordinated expression of main CsHPL (TEA008699), CsAOS (TEA001041), and CsJMT (TEA015791) control the flow of lipids degradation and MeJA production. A strong infected reduction of a key lipoxygenase gene, CsLOX6 (TEA009423), in tea buds significantly reduced the level of jasmonates and expression of downstream genes, accompanied by SA, MeSA level rising, and ABA declining. We have identified a key CsLOX6, as well as established galactolipids, mainly 34:3/36:6/36:3 DGDG and 36:6/36:5/36:4 MGDG, sources for MeJA biosynthesis regulated by dynamics hormone and controlled by coordinated expressed CsHPL (TEA008699), CsAOS (TEA001041), and CsJMT (TEA015791). Our findings provide a theoretical basis for breeding high-quality black tea and offer valuable insights for improving processing methods.

Copper increases laccase gene transcription and extracellular laccase activity in Pleurotus eryngii KS004.

The white-rot fungus Pleurotus eryngii secretes various laccases involved in the degradation of a wide range of chemical compounds. Since the laccase production is relatively low in fungi, many efforts have been focused on finding ways to increase it, so in this study, we investigated the effect of copper on the transcription of the pel3 laccase gene and extracellular laccase activity. The results indicate that adding 0.5 to 2 mM copper to liquid cultures of P. eryngii KS004 increased both pel3 gene transcription and extracellular laccase activity in a concentration-dependent manner. The most significant increase in enzyme activity occurred at 1 mM Cu2+, where the peak activity was 4.6 times higher than in control flasks. Copper also induced the transcription of the laccase gene pel3. The addition of 1.5 and 2 mM Cu2+ to fungal culture media elevated pel3 transcript levels to more than 13-fold, although the rate of induction slowed down at Cu2+ concentrations higher than 1.5 mM. Our findings suggest that copper acts as an inducer in the regulation of laccase gene expression in P. eryngii KS004. Despite its inhibitory effect on fungal growth, supplementing cultures with copper can lead to an increased extracellular laccase production in P. eryngii.

Homozygous HOXC13 Variant Causes Pure Hair and Nail Ectodermal Dysplasia via Reduction in Protein Stability

Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder characterized by reduced or absent hair and dystrophic nails. PHNED is caused by pathogenic variants in genes involved in hair and nail development, including HOXC13. Previously reported biallelic HOXC13 pathogenic variants led to PHNED by either disrupting protein expression through nonsense‐mediated decay or altering the DNA‐binding affinity of the homeobox domain of HOXC13. Here, we report a case of HOXC13‐related PHNED with a rare homozygous variant, c.931C>T, p.Arg311Trp. Similarly to previously reported missense variants, p.Arg311Trp resides in the homeobox domain of HOXC13 and was assumed to lead to the decreased transcriptional activity of target genes. However, in contrast with previously reported variants, in vitro overexpression assays revealed that the p.Arg311Trp variant decreases HOXC13 protein stability, which is corroborated by a series of in silico predictions. Computational models further suggest that p.Arg311Trp results in a structural rearrangement with loss of interhelical connection between Arg311 in α‐helix 3 and Glu276 in α‐helix 1. Altogether, our results suggest a novel molecular mechanism causative of PHNED, whereby biallelic pathogenic variants in HOXC13 may result in decreased protein stability and consequently decreased transcriptional activity of target genes essential for hair and nail development.

Transcriptional landscape of the pMP7017 megaplasmid and its impact on the Bifidobacterium breve UCC2003 transcriptome.

The 190 kb megaplasmid pMP7017 of Bifidobacterium breve JCM7017 represents the first conjugative and largest plasmid characterised within this genus to date. In the current study, we adopted an integrated approach combining transcriptomics, whole genome comparative analysis and metagenomic data mining to understand the biology of pMP7017 and related megaplasmids, and to assess the impact of plasmid-carriage on the host strain. The data generated revealed variations within basic features of promoter elements which correlate with a high level of transcription on the plasmid and highlight the transcriptional activity of genes encoding both offensive and defensive adaptations, including a Type IIL restriction-modification system, an anti-restriction system and four Type II toxin-antitoxin systems. Furthermore, a highly transcribed tmRNA, which likely provides translational support to the host strain, was identified, making pMP7017 the first plasmid of the Bifidobacterium genus and the smallest plasmid known to express a tmRNA. Analyses of synteny and variability among pMP7017 and related plasmids indicate substantial diversity in gene organisation and accessory gene cargo highlighting diverse (co-)evolution and potential host-specific rearrangements and adaptations. Systematic analysis of the codon usage profile of transcriptionally active pMP7017-encoded genes suggests that pMP7017 originated from (sub)species of Bifidobacterium longum. Furthermore, mining of metagenomic data suggests the presence of pMP7017-homologues in ~10% of microbiome samples, mostly infants and/or mothers from various geographical locations. Comparative transcriptome analysis of the B. breve UCC2003 chromosome in the presence or absence of pMP7017 revealed differential expression of genes representing 8% of the total gene pool. Genes involved in genetic information processing were exclusively upregulated, while altered expression of genes involved in biofilm production and polysaccharide biosynthesis was also observed.