Gene Regulatory Factors Research Articles

Dietary manganese nano-particles improves gene regulation and biochemical attributes for mitigation of lead and ammonia toxicity in fish

In the present study, we explored the capability of manganese nanoparticles (Mn-NPs) to alleviate the toxicity induced by lead (Pb) and ammonia (NH3) toxicity in Oreochromis niloticus (GIFT strain). The experiment followed a completely randomized design, including a control group (Mn-NPs-0 mg kg−1 diet) and groups exposed to Pb and NH3 alongwith Mn-NPs at 2 and 3 mg kg−1. Cortisol levels were significantly elevated in Pb + NH3 group whereas reduced by Mn-NPs diets. Gene expressions of HSP 70, iNOS, CYP 450, and Cas 3a were notably upregulated by Pb + NH3 group and downregulated by Mn-NPs diets. The cellular metabolic enzymes were affected by Pb + NH3 exposure and mitigated by Mn-NPs diets. The liver and kidney exhibited reduced activities of catalase, superoxide dismutase, and glutathione-s-transferase with Mn-NPs diets. Concurrently, immune-related genes such as total immunoglobulin (Ig) and tumor necrosis factor (TNFα) were upregulated in the Mn-NPs-fed groups. Growth performance indicators, including weight gain %, feed conversion ratio, specific growth rate, protein efficiency ratio, and relative feed intake were adversely affected by Pb + NH3 stress but improvement with Mn-NPs diets. Genes associated with growth performance, such as growth hormone (GH), growth hormone regulatory (GHR1), and myostatin, exhibited enhancements in response to Mn-NPs diets. Digestive enzymes, including protease and amylase were also enhanced by Mn-NPs diets. Additionally, Mn-NPs diets led to a reduction in the bioaccumulation of lead. This study aims to investigate the role of Mn-NPs in mitigating the effects of lead and ammonia toxicity on fish by examining various biochemical and gene regulatory factors to enhance fish wellbeing.

Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors

Alternative splicing significantly expands biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental and tissue-dependent alternative exons often control protein-protein interactions; yet, only a minor fraction of these events have been characterized. Using affinity purification-mass spectrometry (AP-MS), we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners, which in some cases form unexpected links with coupled processes. Notably, a neural exon in Chtop regulates its interaction with the Prmt1 methyltransferase and DExD-Box helicases Ddx39b/a, affecting its methylation and activity in promoting RNA export. Additionally, a neural exon in Sap30bp affects interactions with RNA processing factors, modulating a critical function of Sap30bp in promoting the splicing of <100 nt "mini-introns" that control nuclear RNA levels. AP-MS is thus a powerful approach for elucidating the multifaceted functions of proteins imparted by context-dependent alternative exons.

Nuclear Condensates Are a Therapeutic Vulnerability in NPM1-Mutant Acute Myeloid Leukemia

The development of targeted therapies against drivers of malignancy requires a robust mechanistic understanding of the pathogenic axis underlying disease. Several subtypes of acute myeloid leukemias (AML), such as those driven by mutations in IDH1/2, FLT3, and KMT2A, have benefitted from the development of novel therapeutics that disrupt their transcriptional program. In contrast, there are no approved therapies that target the mutant version of Nucleophosmin (NPM1), which is the most common driver in adult AML. Mutations in NPM1 result in export of the mutant protein (NPM1c) to the cytoplasm, prompting the suggestion that aberrant localization leads to neomorphic and pathogenic functions of NPM1c in the cytoplasm. In parallel, recent data from several groups has suggested that NPM1c directly regulates gene expression within the nucleus. Despite this progress, a well-defined mechanism for NPM1c-mediated gene regulation is lacking, hindering the development of NPM1c-targeted therapies. To investigate the function of NPM1c, we generated over a dozen AML cell lines expressing unique NPM1c truncations and characterized their impact on the leukemic transcriptome and cell morphology. To our surprise, we found that although the majority of these truncated proteins reside in the cytoplasm, most were unable to recapitulate the transcriptional program driven by full-length NPM1c, and cells subsequently underwent differentiation and growth arrest. We further explored the chromatin binding ability of these truncated proteins to key genomic regions such as the HOXA cluster and MEIS1 through CUT&amp;RUN analysis. Next, we identified a region of the NPM1c protein critical for active nuclear import (NLS). Strikingly, disruption of this region enforces cytoplasmic sequestration of the truncated NPM1c protein. However, cells harboring this truncation fail to maintain leukemic gene expression and differentiate. Together these results suggest that aberrant localization and/or shuttling of NPM1c between cellular compartments is insufficient to maintain leukemic gene expression and cell-stemness. We next considered whether NPM1c had a de-novo function that may explain its role in gene regulation. We have recently shown that NPM1c forms phase-separated condensates in the nucleus that regulate HOX gene expression and are necessary for stem-ness. These NPM1c condensates recruit multiple gene regulatory factors including XPO1, NUP98, and KMT2A (Figure 1). Through selective degradation of NPM1c, or pharmacological inhibition of XPO1, we found that disruption of nuclear condensates abrogates recruitment of these protein co-factors. We further explore the effect of drug compounds known to inhibit NPM1-mutant AML on condensate behavior and chromatin binding. This condensate behavior is a defining feature of heterotypic phase separation, a process in which two or more biomolecules form multivalent interactions to create a condensate. Importantly, we found that significant overexpression of NPM1c causes an imbalance in the ratio between NPM1c and its binding partners, disrupting normal condensate formation, and causing subsequent differentiation and growth arrest. Collectively, these results suggest that therapeutic targeting of NPM1c, or a host of interacting partners, may disrupt condensate formation and the leukemic transcriptional program, providing an exciting new therapeutic vulnerability in NPM1-mutant AML.

Dual analysis of wild-type and attenuated Orf virus and host cell transcriptomes revealed novel virus-host cell interactions.

Currently, the only available commercial vaccines for Orf virus (ORFV) are live attenuated vaccines, which present a potential risk of reversion to virulence. Therefore, understanding the pathogenic mechanisms of different virulent strains of ORFV and host immune responses triggered by these viruses is crucial for developing new vaccines and interventions. In this study, we found that the attenuated strain downregulates the host innate immune response and antiviral activity. In addition, we noted that the wild-type strain can induce the immune response pattern centered on interferon-stimulated genes and interferon regulatory factor gene family. We predicted that STAT1 and STAT2 are the main transcription factors upstream of target gene promoters through gene regulatory networks and exert significant regulatory effects on co-expressed genes. Our study elucidated the complex interaction between ORFV strains and host cell immune responses, providing new insights into vaccine research for ORFV.

Topologically Overlapped Fused LASSO Measure for Reconstructing Gene Regulation Networks

Identifying target genes in Gene Regulation Network (GRN) models has always been challenging in Systems Biology. In this regard, indirect gene regulatory hierarchical architectures may be promising enough, considering varied topological structures and unknown gene regulation factors. Such causal regulations can be investigated, keeping in force all perturbation experiments of a dataset. Contemporary research primarily highlights direct interaction networks, which mostly forego the inevitable presence of a third entity, if any, towards varied forms of causal regulations. In this article, we have developed a hierarchical algorithm that unveils the genetic wiring through the Fused Least Absolute Shrinkage and Selection Operator (Fused-LASSO) technique with a Topological Overlap (TO) measure as the interaction structure. The potential power of this proposed model is studied over YEAST Cell Cycle, and Human cancer cell line data (HeLa). In this connection, the different statistically significant hierarchical regulation outcomes maintaining parity with the direct interaction structures, if any, to the target genes may throw new light on gene regulation statistics.

Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3’-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1’s abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications.

Abstract 18042: Induction of Cardiac Reprogramming Following Injury Through Delivery of a Single Factor, PHF7

Direct reprogramming of resident cardiac fibroblasts into cardiomyocytes is an attractive strategy to restore cardiac function following myocardial infarction (MI). However, canonical and extensively studied reprogramming factors (e.g. Gata4, Mef2c, Tbx5) have shown to be ineffective in reprogramming adult human fibroblasts. Furthermore, current human cocktails require an excess of factors, limiting the clinical translation and safety of this technology. Recently, we undertook a large screen of gene regulatory factors and identified the epigenetic factor PHF7 as the most potent activating factor of adult human fibroblast reprogramming, and described its ability to bind and modify chromatin accessibility at cardiac super enhancers. In this manuscript, we describe the ability of PHF7 to activate adult reprogramming in vitro in absence of canonical reprogramming factors, with PHF7, MEF2C, and TBX5 (PMT) or PHF7 and TBX5 (PT) alone. These minimalist PHF7 cocktails induced broad and unique cardiac transcriptomes in comparison to canonical cocktails. Importantly, when overexpressed in isolation in fibroblasts, PHF7 strongly activated expression of several cardiac transcription factors, establishing its role as a cardiac master regulator. When delivered to the mouse heart following MI, minimalist PHF7 cocktails were found to significantly recover cardiac function and decrease fibrotic scar formation. Interestingly, delivery of PHF7 alone following MI was sufficient to improve cardiac function and induce remodeling through activation of bona fide reprogramming events in vivo as determined by genetic tracing of the Postn MCM lineage. Here, we report for the first time the ability of a single reprogramming factor, PHF7, to induce fibroblast to cardiomyocyte reprogramming and recover cardiac function following injury in vivo.

The STING-IRF3 Signaling Pathway, Mediated by Endoplasmic Reticulum Stress, Contributes to Impaired Myocardial Autophagic Flux After Ischemia/Reperfusion.

This study aimed to determine whether endoplasmic reticulum (ER) stress is involved in impaired autophagy after myocardial ischemia/reperfusion (M-I/R) and elucidate the underlying mechanisms. The expression levels of stimulator of interferon gene (STING) and interferon regulatory transcription factor 3 (IRF3) phosphorylation increased in M-I/R heart tissues and hypoxia-treated/reoxygenation-treated H9c2 cells. The ER stress inhibitor 4-phenylbutyric acid (4-PBA) significantly suppressed the stimulation of STING-IRF3 transcription and alleviated cardiac dysfunction caused by M-I/R injury. In addition, 4-PBA reversed ischemia-induced/reperfusion-induced autophagic flux dysfunction, as demonstrated by a decrease in p 62 and LC3 levels. Similarly, the protective effect of STING deficiency on myocardial cell damage was achieved by the recovery of autophagic flux. Conversely, the protective effect of 4-PBA against hypoxia/reoxygenation injury in cardiomyocytes was offset by STING overexpression, wherein the activated STING-IRF3 pathway promoted the expression of Rubicon (a negatively-regulated autophagic molecule) by binding to the Rubicon promoter. Rubicon ablation effectively counteracts the adverse effects of STING overexpression in cardiomyocytes. The data showed that STING-IRF3 signaling of ER stress receptors is particularly important in the progression of physiological M-I/R caused by the inhibition of autophagic flow in vivo and in vitro.

Single-cell analysis of prenatal and postnatal human cortical development.

We analyzed >700,000 single-nucleus RNA sequencing profiles from 106 donors during prenatal and postnatal developmental stages and identified lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types, and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineated enhancer gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identified the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs up-regulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development.

THU488 Genetic Alterations In CREBRF Influence Prostate Cancer Survival And Impact Prostate Tissue Homeostasis In Mice

Abstract Disclosure: L.E. Pascal: None. K.A. Frahm: None. K. Skalitzky: None. D.B. DeFranco: None. L. Rigatti: None. R. Lu: None. T. Liu: None. Background: Risk factors for prostate cancer include age, environmental factors, race and ethnicity. Genetic variants in cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) gene are frequently observed in Pacific Islanders, a population with elevated prostate cancer incidence. CREBRF has been shown to play a role in other cancers, however its function in prostate homeostasis and tumorigenesis has not been previously explored. Methods: Alterations in CREBRF were identified in prostate cancer patients via in silico analysis of several publicly available datasets through cBioPortal. Male Crebrf knockout mice were generated and examined for prostate defects at 4 months of age. Immunohistochemical staining of murine prostate sections was used to determine the impact of Crebrf knockout on proliferation, apoptosis, inflammation and blood vessel density in the prostate. Serum adipokine levels were measured using a Luminex Multiplex Assay. Results: CREBRF alterations were identified in up to 4.05% of prostate tumors and the mutations identified were categorized as likely damaging. Median survival of patients with CREBRF alterations was 41 months, compared to 131 months for patients without CREBRF alterations. In the murine model, the prostates of Crebrf knockout mice had reduced epithelial proliferation and increased TUNEL+ apoptotic cells. Circulating adipokines PAI-1 and MCP-1 were also altered in Crebrf knockout mice compared to age-matched controls. Conclusions: Prostate cancer patients with CREBRF alterations were associated with decreased overall survival suggesting that CREBRF may play a role in prostate tumorigenesis and progression. The murine knockout model demonstrated that CREBRF could modulate proliferation and apoptosis and macrophage density in the prostate. Serum levels of adipokines PAI-1 and MCP-1 were also altered and could contribute to the phenotypic changes observed in the prostates of Crebrf knockout mice. Future studies focused on populations susceptible to CREBRF mutations and mechanistic studies will be required to fully elucidate the potential role of CREBRF in prostate tumorigenesis. Presentation: Thursday, June 15, 2023

The ability to induce heat shock transcription factor-regulated genes in response to lethal heat stress is associated with thermotolerance in tomato cultivars.

Heat stress is a severe challenge for plant production, and the use of thermotolerant cultivars is critical to ensure stable production in high-temperature-prone environments. However, the selection of thermotolerant cultivars is difficult due to the complex nature of heat stress and the time and space needed for evaluation. In this study, we characterized genome-wide differences in gene expression between thermotolerant and thermosensitive tomato cultivars and examined the possibility of selecting gene expression markers to estimate thermotolerance among different tomato cultivars. We selected one thermotolerant and one thermosensitive cultivar based on physiological evaluations and compared heat-responsive gene expression in these cultivars under stepwise heat stress and acute heat shock conditions. Transcriptomic analyses reveled that two heat-inducible gene expression pathways, controlled by the heat shock element (HSE) and the evening element (EE), respectively, presented different responses depending on heat stress conditions. HSE-regulated gene expression was induced under both conditions, while EE-regulated gene expression was only induced under gradual heat stress conditions in both cultivars. Furthermore, HSE-regulated genes showed higher expression in the thermotolerant cultivar than the sensitive cultivar under acute heat shock conditions. Then, candidate expression biomarker genes were selected based on the transcriptome data, and the usefulness of these candidate genes was validated in five cultivars. This study shows that the thermotolerance of tomato is correlated with its ability to maintain the heat shock response (HSR) under acute severe heat shock conditions. Furthermore, it raises the possibility that the robustness of the HSR under severe heat stress can be used as an indicator to evaluate the thermotolerance of crop cultivars.

A negative correlation between hsa-miR29a-3p level and HIV-1 viral load in human serum; potentiate criteria for patients screening

Human Immunodeficiency Virus type-1 (HIV-1) causes persistent and life-threatening infection, leading to progressive disease. MicroRNAs (miRNAs) are regulators of gene expression which can be found in circulating human blood samples. hsa-miR-29a-3p has been identified as a potential regulator of the Negative Regulatory Factor (Nef) gene from the HIV-1 viral genome. In this study, we aimed to compare the serum levels of hsa-miR-29a-3p with HIV-1 viral load in a substantial number of infected individuals. We collected serum samples from a total of 48 participants, including 36 untreated HIV-positive patients, and 12 HIV-negative individuals as a control group, matched for age and sex. The HIV-1 viral load in both the case and control groups was confirmed using qRT-PCR. Subsequent qRT-PCR analysis of circulating hsa-miR-29a-3p levels revealed lower miRNA expression in the groups with higher viral loads. A negative correlation (r = −0.58) was calculated between hsa-miR-29a-3p levels and HIV-1 viral load. These findings suggest that the expression level of hsa-miR-29a-3p may serve as an indicator of HIV-1 viral load in human serum samples. Additionally, this miR may hold promise as a potential tool for enhancing HIV-1 treatment strategies.

Abstract P1123: Phf7 As A Critical Driver Of Adult Cardiac Reprogramming

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Further, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 is the first epigenetic factor shown to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

Shared Gene Targets of the ATF4 and p53 Transcriptional Networks.

The master tumor suppressor p53 regulates multiple cell fate decisions, such as cell cycle arrest and apoptosis, via transcriptional control of a broad gene network. Dysfunction in the p53 network is common in cancer, often through mutations that inactivate p53 or other members of the pathway. Induction of tumor-specific cell death by restoration of p53 activity without off-target effects has gained significant interest in the field. In this study, we explore the gene regulatory mechanisms underlying a putative anticancer strategy involving stimulation of the p53-independent integrated stress response (ISR). Our data demonstrate the p53 and ISR pathways converge to independently regulate common metabolic and proapoptotic genes. We investigated the architecture of multiple gene regulatory elements bound by p53 and the ISR effector ATF4 controlling this shared regulation. We identified additional key transcription factors that control basal and stress-induced regulation of these shared p53 and ATF4 target genes. Thus, our results provide significant new molecular and genetic insight into gene regulatory networks and transcription factors that are the target of numerous antitumor therapies.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

Evaluation of the Possible Pathways Involved in the Protective Effects of Quercetin, Naringenin, and Rutin at the Gene, Protein and miRNA Levels Using In-Silico Multidimensional Data Analysis.

Flavonoids are secondary metabolites that are non-essential for plant growth or survival, and they also provide numerous health benefits to humans. They are antioxidants that shield plants from the ill effects of ultraviolet light, pests, and diseases. They are beneficial to health for several reasons, including lowering inflammation, boosting cardiovascular health, and lowering cancer risk. This study looked into the physicochemical features of these substances to determine the potential pharmacological pathways involved in their protective actions. Potential targets responsible for the protective effects of quercetin, naringenin, and rutin were identified with SwissADME. The associated biological processes and protein-protein networks were analyzed by using the GeneMANIA, Metascape, and STRING servers. All the flavonoids were predicted to be orally bioavailable, with more than 90% targets as enzymes, including kinases and lyases, and with common targets such as NOS2, CASP3, CASP9, CAT, BCL2, TNF, and HMOX1. TNF was shown to be a major target in over 250 interactions. To extract the "biological meanings" from the MCODE networks' constituent parts, a GO enrichment analysis was performed on each one. The most important transcription factors in gene regulation were RELA, NFKB1, PPARG, and SP1. Treatment with quercetin, naringenin, or rutin increased the expression and interaction of the microRNAs' hsa-miR-34a-5p, hsa-miR-30b-5p, hsa-let-7a-5p, and hsa-miR-26a-1-3p. The anticancer effects of hsa-miR-34a-5p have been experimentally confirmed. It also plays a critical role in controlling other cancer-related processes such as cell proliferation, apoptosis, EMT, and metastasis. This study's findings might lead to a deeper comprehension of the mechanisms responsible for flavonoids' protective effects and could present new avenues for exploration.

MOSR and NDHA Genes Comprising G-Quadruplex as Promising Therapeutic Targets against Mycobacterium tuberculosis: Molecular Recognition by Mitoxantrone Suppresses Replication and Gene Regulation.

Occurrence of non-canonical G-quadruplex (G4) DNA structures in the genome have been recognized as key factors in gene regulation and several other cellular processes. The mosR and ndhA genes involved in pathways of oxidation sensing regulation and ATP generation, respectively, make Mycobacterium tuberculosis (Mtb) bacteria responsible for oxidative stress inside host macrophage cells. Circular Dichroism spectra demonstrate stable hybrid G4 DNA conformations of mosR/ndhA DNA sequences. Real-time binding of mitoxantrone to G4 DNA with an affinity constant ~105-107 M-1, leads to hypochromism with a red shift of ~18 nm, followed by hyperchromism in the absorption spectra. The corresponding fluorescence is quenched with a red shift ~15 nm followed by an increase in intensity. A change in conformation of the G4 DNA accompanies the formation of multiple stoichiometric complexes with a dual binding mode. The external binding of mitoxantrone with a partial stacking with G-quartets and/or groove binding induces significant thermal stabilization, ~20-29 °C in ndhA/mosR G4 DNA. The interaction leads to a two/four-fold downregulation of transcriptomes of mosR/ndhA genes apart from the suppression of DNA replication by Taq polymerase enzyme, establishing the role of mitoxantrone in targeting G4 DNA, as an alternate strategy for effective anti-tuberculosis action in view of deadly multi-drug resistant tuberculosis disease causing bacterial strains t that arise from existing therapeutic treatments.

Positive and Relaxed Selective Pressures Have Both Strongly Influenced the Evolution of Cryonotothenioid Fishes during Their Radiation in the Freezing Southern Ocean.

Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. By identifying the genomic signatures of selection, this study aims to identify the functional classes of genes that have been changed following two major physiological transitions: the onset of freezing temperatures and the loss of hemoproteins. Looking at the changes that followed the onset of freezing temperatures, positive selective pressure was found among a set of broadly acting gene regulatory factors, suggesting a route through which cryonotothenioid gene expression has been retooled for life in the cold. Further, genes related to the cell cycle and cellular adhesion were found under positive selection suggesting that both present key challenges to life in freezing waters. By contrast, genes showing signatures of the relaxation of selective pressure showed a narrower biological impact, acting on genes related to mitochondrial function. Finally, although chronic cold-water temperatures appear correlated with substantial genetic change, the loss of hemoproteins resulted in little observable change in protein-coding genes relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection shows that long-term exposure to cold has led to profound changes in cryonotothenioid genomes that may make it challenging for them to adapt to a rapidly changing climate.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

DNA repair byproduct 8-oxoguanine base promotes myoblast differentiation

Muscle contraction increases the level of reactive oxygen species (ROS), which has been acknowledged as key signaling entities in muscle remodeling and to underlie the healthy adaptation of skeletal muscle. ROS inevitably endows damage to various cellular molecules including DNA. DNA damage ought to be repaired to ensure genome integrity; yet, how DNA repair byproducts affect muscle adaptation remains elusive. Here, we showed that exercise elicited the generation of 8-oxo-7,8-dihydroguanine (8-oxoG), that was primarily found in mitochondrial genome of myofibers. Upon exercise, TA muscle's 8-oxoG excision capacity markedly enhanced, and in the interstitial fluid of TA muscle from the post-exercise mice, the level of free 8-oxoG base was significantly increased. Addition of 8-oxoG to myoblasts triggered myogenic differentiation via activating Ras-MEK-MyoD signal axis. 8-Oxoguanine DNA glycosylase1 (OGG1) silencing from cells or Ogg1 KO from mice decreased Ras activation, ERK phosphorylation, MyoD transcriptional activation, myogenic regulatory factors gene (MRFs) expression. In reconstruction experiments, exogenously added 8-oxoG base enhanced the expression of MRFs and accelerated the recovery of the injured skeletal muscle. Collectively, these data not only suggest that DNA repair metabolite 8-oxoG function as a signal entity for muscle remodeling and contribute to exercise-induced adaptation of skeletal muscle, but also raised the potential for utilizing 8-oxoG in clinical treatment to skeletal muscle damage-related disorders.