Nuclear Localization Research Articles

E3 Ubiquitin Ligase Ring Finger Protein 2 Alleviates Cerebral Ischemia-Reperfusion Injury by Stabilizing Mesencephalic Astrocyte-Derived Neurotrophic Factor Through Monoubiquitination.

Cerebral ischemic stroke (IS) is one of the leading causes of morbidity and mortality globally. However, the mechanisms underlying IS injury remain poorly understood. Ring finger protein 2 (RNF2), the member of the polycomb family (PcG), has been implicated in diverse biological and pathological conditions. However, whether RNF2 plays a role in IS progression is not clarified. This study aims to investigate the potential effects of RNF2 on IS. The effects of RNF2 were studied in human postmortem IS brains, a rat model of IS, tunicamycin (TM)-induced mouse neuroblastoma neuro2a (N2a) cells, and oxygen-glucose deprivation/reperfusion (OGD/R)-induced SH-SY5Y cells. Here, we demonstrated that RNF2 was markedly upregulated both in human postmortem IS brains and ischemic rat brains and RNF2 overexpression alleviated brain injury induced by middle cerebral artery occlusion by reducing neuron apoptosis. Mechanistically, we found that RNF2 is an E3 ubiquitin ligase for the mesencephalic astrocyte-derived neurotrophic factor (MANF), which confers protection against brain ischemia. RNF2 interacted with MANF and promoted the monoubiquitination of MANF, consequently facilitating its stability and nuclear localization. Collectively, RNF2 is identified as a critical inhibitor of IS injury by stabilizing MANF through monoubiquitination, suggesting that RNF2 is a potential therapeutic target for IS.

TOWARDS THE DESIGN OF LIGANDS OF THE INTERNAL POCKET TEADS C-TERMINAL DOMAIN

The Hippo pathway controls in organ size and tissue homeostasis through regulating cell growth, proliferation and apoptosis. Phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) regulates their nuclear import and therefore their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several solid cancers making YAP/TAZ-TEAD interaction a new anti-cancer target. We identified by screening a small in-house library, 5-benzyloxindole which binds to hTEAD2 at its internal/palmitate pocket. Its optimization led to covalent inhibitors bearing different warhead. Soaking with hTEAD2 gave seven new crystal structures where the ligands occupied palmitate pocket. 5-Benzyloxyindoles armed with vinylsulfamide moiety inhibit YAP/TAZ-TEAD target genes expression and breast cancer cell proliferation at micromolar concentration.

Modulating versatile pathways using a cleavable PEG shell and EGFR-targeted nanoparticles to deliver CRISPR-Cas9 and docetaxel for triple-negative breast cancer inhibition.

Human antigen R (HuR), an RNA-binding protein, is implicated in regulating mRNA stability and translation in cancer, especially in triple-negative breast cancer (TNBC), a highly aggressive form. CRISPR/Cas9-mediated HuR knockout (HuR CRISPR) presents a promising genetic therapeutic approach, but it encounters transfection limitations. Docetaxel (DTX), an effective cytotoxic agent against metastatic breast cancer (BC), faces challenges related to vehicle-associated adverse events in DTX formulations. Therefore, we designed multifunctional nanoparticles with pH-sensitive PEG derivatives and targeting peptides to enable efficient HuR CRISPR and DTX delivery to human TNBC MDA-MB-231 cells and tumor-bearing mice. Our findings indicated that these nanoparticles displayed pH-responsive cytotoxicity, precise EGFR targeting, efficient tumor penetration, successful endosomal escape, and accurate nuclear and cytoplasmic localization. They also demonstrated the ability to spare normal cells and prevent hemolysis. Our study concurrently modulated multiple pathways, including EGFR, Wnt/β-catenin, MDR, and EMT, through the regulation of EGFR/PI3K/AKT, HuR/galectin-3/GSK-3β/β-catenin, and P-gp/MRPs/BCRP, as well as YAP1/TGF-β/ZEB1/Slug/MMPs. The combined treatment arrested the cell cycle at the G2 phase and inhibited EMT, effectively impeding tumor progression. Tissue distribution, biochemical assays, and histological staining revealed the enhanced safety profile of pH-responsive PEG- and peptide-modified nanoformulations in TNBC mice. The DTX-embedded and peptide-modified nanoparticles mitigated the side effects of DTX, enhanced cytotoxicity in TNBC MDA-MB-231 cells, and exhibited remarkable antitumor efficacy and safety in TNBC-bearing mice with HuR CRISPR deletion. Collectively, the combination therapy of DTX and CRISPR/Cas9 offers an effective platform for delivering antineoplastic agents and gene-editing systems to combat tumor resistance and progression in TNBC.

Ethylene Response Transcription Factor 5 (ERF5) Enhances Defense Against Tobacco Curly Shoot Virus and Associated Betasatellite (TbCSV/TbCSB) in Nicotiana benthamiana

Begomovirus/betasatellite disease complex significantly threatens global crop production. Identifying potential plant antiviral genes is crucial for disease control. Nicotiana benthamiana is susceptible to viruses and contains 266 ethylene response transcription factors (ERFs). This study identified 29 NbERFs that were differentially upregulated in tobacco curly shoot virus and its associated betasatellite (TbCSV/TbCSB) infection, with ERF5 being the most common. Nine NbERF5s cluster phylogenetically and Niben101Scf00163g22002 (NbERF5) responds significantly to exogenous ACC treatment. Further analysis confirms the nuclear localization and transcriptional activation activity of NbERF5. Protein interaction assays demonstrate that NbERF5 has no self-interaction and does not interact with the βC1 protein of TbCSB. Silencing NbERF5 enhances TbCSV/TbCSB infection, and overexpression of NbERF5 inhibits TbCSV/TbCSB infection. Importantly, NbERF5 positively regulates the expression of the pathogenesis-related (PR) genes, NbPR1a and NbNPR1. Our findings suggest that NbERF5 enhances TbCSV/TbCSB resistance by activating the PR genes, indicating that NbERF5 is a potential antiviral gene.

Dephosphorylation of bZIP59 by PP2A ensures appropriate shade avoidance response in Arabidopsis.

Changes in light quality and quantity experienced by many shade-intolerant plants grown in close proximity lead to transcriptional reprogramming and shade avoidance syndrome (SAS). Despite the importance of phosphorylation-dependent signaling in cellular physiology, phosphorylation events during SAS are largely unknown. Here, we examined shade-regulated phosphorylation events in Arabidopsis using quantitative phosphoproteomics. We confirmed shade-induced dephosphorylation of bZIP59, a basic region/leucine zipper motif (bZIP) transcription factor. Shade treatment promotes the nuclear localization of bZIP59, which can be mimicked by mutation of the phosphorylation sites on bZIP59. Phenotypic analysis identified that bZIP59 negatively regulated shade-induced hypocotyl elongation. bZIP59 repressed the shade-induced activation of certain growth-related genes, while shade increased the DNA binding of bZIP59. Furthermore, the protein phosphatase 2A (PP2A) mediated dephosphorylation of bZIP59. Our study characterized a previously unidentified mechanism by which the phytochrome B (phyB)-PP2A-bZIP59 regulatory module integrates shade signals and transcriptomes, broadening our knowledge of phosphorylation strategies for rapid adaptation to shade.

Oncogenic KRAS Mutations Confer a Unique Mechanotransduction Response to Peristalsis in Colorectal Cancer Cells.

Colorectal cancer (CRC) tumors start as polyps on the inner lining of the colorectum, where they are exposed to the mechanics of peristalsis. Our previous work leveraged a custom-built peristalsis bioreactor to demonstrate that colonic peristalsis led to cancer stem cell enrichment in CRC cells. However, this malignant mechanotransductive response was confined to select CRC lines that harbored an oncogenic mutation in the KRAS gene. Here, we explored the involvement of activating KRAS mutations on peristalsis-associated mechanotransduction in CRC. Peristalsis enriched cancer stem cell marker LGR5 in KRAS mutant lines, in a Wnt-ligand-independent manner. Conversely, LGR5 enrichment in wild type KRAS lines exposed to peristalsis were minimal. LGR5 enrichment downstream of peristalsis translated to increased tumorigenicity in vivo. Differences in mechanotransduction was apparent via unbiased gene set enrichment analysis, where many unique pathways were enriched in wild type vs. mutant lines. Peristalsis also triggered β-catenin nuclear localization independent of Wnt-ligands, particularly in KRAS mutant lines. The involvement of KRAS was validated via gain and loss of function strategies. Peristalsis induced β-catenin activation and LGR5 enrichment depended on the activation of the MEK/ERK cascade. Taken together, our results demonstrated that oncogenic KRAS mutations conferred a unique peristalsis-associated mechanotransduction response to colorectal cancer cells, resulting in cancer stem cell enrichment and increased tumorigenicity. These mechanosensory connections can be leveraged in improving the sensitivity of emerging therapies that target oncogenic KRAS. Implications: Oncogenic KRAS empowers colorectal cancer cells to harness the mechanics of colonic peristalsis for malignant gain, independent of other cooperating signals. .

Regulation of TAR DNA binding protein 43 (TDP-43) homeostasis by cytosolic DNA accumulation

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein predominantly localized in the nucleus under physiological conditions. TDP-43 proteinopathy, characterized by cytoplasmic aggregation and nuclear loss, is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Thus it is crucial to understand the molecular mechanism regulating TDP-43 homeostasis. Here, we show that the uptake of oligodeoxynucleotides (ODNs) induces reversible TDP-43 cytoplasmic puncta formation in both neurons and glia and ODNs facilitate the liquid-liquid phase separation of TDP-43 in vitro. Importantly, persistent accumulation of DNA in the cytoplasm leads to nuclear depletion of TDP-43 and enhanced production of a short isoform of TDP-43 (sTDP-43). In addition, in response to ODN uptake, the nuclear import receptor karyopherin subunit β1 (KPNB1) is sequestered in the cytosolic TDP-43 puncta. ALS-linked Q331K mutation decreases the dynamics of cytoplasmic TDP-43 puncta and increases the levels of sTDP-43. Moreover, the TDP-43 cytoplasmic puncta are induced by DNA damage and by impaired nuclear envelope integrity due to Lamin A/C deficiency. In summary, our data support that abnormal DNA accumulation in the cytoplasm may be one of the key mechanisms leading to TDP-43 proteinopathy and provides novel insights into molecular mechanisms of ALS caused by TDP-43 mutations.

Sodium Benzoate Induces Fat Accumulation and Reduces Lifespan via the SKN-1/Nrf2 Signaling Pathway: Evidence from the Caenorhabditis elegans Model.

Sodium benzoate (SB) is widely used in food products, cosmetics, and medical solutions due to its antimicrobial properties. While it is generally considered safe and has potential neuroprotective benefits, SB has also been linked to adverse effects, including hepatic oxidative stress and inflammation. However, the potential effects of SB on obesity and lifespan remain poorly understood. In this study, we investigated the effects of SB on fat accumulation and lifespan using the nematode Caenorhabditis elegans (C. elegans) as a model system. Wild-type worms were exposed to various SB concentrations (0%, 0.0004%, 0.0008%, 0.004%, and 0.1%) and 0.016% glucose as a positive control for 72 h in liquid or on NGM agar plates. Fat accumulation was assessed through the Oil Red O staining, which revealed that SB induced more fat accumulation compared to vehicle control, even at low concentrations, including the dosage of 0.0004%. Lifespan analysis also demonstrated that SB significantly reduced lifespan in wild-type worms, even at low concentrations. Further investigations found that SKN-1 (an Nrf2 homolog) is necessary for SB-induced fat accumulation and lifespan reduction. Moreover, SB inhibited the nuclear localization of SKN-1 under oxidative stress conditions. These findings suggest that SB may induce fat accumulation and reduce lifespan by inhibiting the oxidative stress-mediated SKN-1 signaling pathway.

Integrin α6β4 Upregulates PTPRZ1 Through UCHL1-Mediated Hif-1α Nuclear Accumulation to Promote Triple-Negative Breast Cancer Cell Invasive Properties.

Integrin α6β4 drives triple-negative breast cancer (TNBC) aggressiveness through the transcriptional regulation of key genes. Here, we investigated how integrin α6β4 regulates protein tyrosine phosphatase receptor type Z1 (PTPRZ1). Using stable re-expression of integrin β4 (ITGB4) in cells naturally devoid of integrin α6β4 or knockdown or knockout (KO) of ITGB4, we found that integrin α6β4 regulates PTPRZ1 expression. To gain mechanistic insight, we focused on Hif-1α due to the impact of integrin α6β4 on a hypoxia-associated signature. We found that nuclear localization of Hif-1α, but not Hif-2α, was substantially enhanced with integrin α6β4 signaling. Hif-1α knockdown by shRNA or chemical inhibition decreased PTPRZ1 expression, while chemical activation of Hif-1α increased it. Upstream of Hif-1α, integrin α6β4 upregulates UCHL1 to stabilize Hif-1α and ultimately regulate PTPRZ1. Inhibition of UCHL1 and PTPRZ1 dramatically decreases integrin α6β4-mediated cell migration and three-dimensional invasive growth. Finally, public breast cancer database analyses demonstrated that ITGB4 correlates with PTPRZ1 and that high expression of ITGB4, UCHL1, HIF1A, and PTPRZ1 associated with decreased overall survival, distant metastasis free survival, post progression survival, and relapse-free survival. In summary, these findings provide a novel function of integrin α6β4 in promoting tumor invasive phenotypes through UCHL1-Hif-1α-mediated regulation of PTPRZ1.

Involvement of Human Cellular Proteins and Structures in Realization of the HIV Life Cycle: A Comprehensive Review, 2024.

Human immunodeficiency virus (HIV) continues to be a global health challenge, with over 38 million people infected by the end of 2022. HIV-1, the predominant strain, primarily targets and depletes CD4+ T cells, leading to immunodeficiency and subsequent vulnerability to opportunistic infections. Despite the progress made in antiretroviral therapy (ART), drug resistance and treatment-related toxicity necessitate novel therapeutic strategies. This review delves into the intricate interplay between HIV-1 and host cellular proteins throughout the viral life cycle, highlighting key host factors that facilitate viral entry, replication, integration, and immune evasion. A focus is placed on actual findings regarding the preintegration complex, nuclear import, and the role of cellular cofactors such as FEZ1, BICD2, and NPC components in viral transport and genome integration. Additionally, the mechanisms of immune evasion via HIV-1 proteins Nef and Vpu, and their interaction with host MHC molecules and interferon signaling pathways, are explored. By examining these host-virus interactions, this review underscores the importance of host-targeted therapies in complementing ART, with a particular emphasis on the potential of genetic research and host protein stability in developing innovative treatments for HIV/AIDS.

Unveiling the functions of the Lim-domain binding protein MaPtaB in Metarhizium acridum.

The Lim-domain binding protein PtaB, a homolog of Mfg1, governs conidiation and biofilm formation in several fungi. PtaB includes a conserved Lim-binding domain and two predicted nuclear localization sequences at its C terminus, and is co-regulated with the transcription factor Som1 downstream of the cyclic AMP-dependent protein kinase A (cAMP/PKA) pathway. However, the function of PtaB in entomopathogenic fungi remain poorly understood. Inactivation of PtaB in Metarhizium acridum resulted in delayed conidial germination, reduced conidial yield and increased sensitivities to cell wall disruptors, ultraviolet B irradiation and heat shock. In addition, the fungal virulence was significantly decreased after deletion of MaPtaB because of impairments in appressorium formation, cuticle penetration and evasion of insect immune responses in M. acridum. The MaPtaB-deletion and MaSom1-deletion strains showed similar phenotypes supporting that MaSom1/MaPtaB complex controls M. acridum normal conidiation and pathogenic progress. Upon loss of MaPtaB or MaSom1, the fungal sporulation mode in M. acridium shifted from microcycle conidiation to normal conidiation on SYA, a microcycle conidiation medium. Transcriptional analysis showed that more differentially expression genes were identified in MaSom1 RNA sequencing, and MaSom1 and MaPtaB may regulate the expression of genes for conidiation, nutrient metabolism and the cell cycle to control conidiation pattern shift. These data corroborate a complex control function for MaPtaB as an important central factor interacting with MaSom1 in the cAMP/PKA pathway, which links stress tolerance, conidiation and virulence in the entomopathogenic fungus M. acridum. © 2024 Society of Chemical Industry.

Progressive polyadenylation and m6A modification of Ighg1 mRNA maintain IgG1 antibody homeostasis in antibody-secreting cells

Antigen-specific antibodies are generated by antibody-secreting cells (ASCs). How RNA post-transcriptional modification affects antibody homeostasis remains unclear. Here, we found that mRNA polyadenylations and N6-methyladenosine (m6A) modifications maintain IgG1 antibody production in ASCs. IgG heavy-chain transcripts (Ighg) possessed a long 3' UTR with m6A sites, targeted by the m6A reader YTHDF1. B cell-specific deficiency of YTHDF1 impaired IgG production upon antigen immunization through reducing Ighg1 mRNA abundance in IgG1+ ASCs. Disrupting either the m6A modification of a nuclear-localized splicing intermediate Ighg1 or the nuclear localization of YTHDF1 reduced Ighg1 transcript stability. Single-cell RNA sequencing identified an ASC subset with excessive YTHDF1 expression in systemic lupus erythematosus patients, which was decreased upon therapy with immunosuppressive drugs. In a lupus mouse model, inhibiting YTHDF1-m6A interactions alleviated symptoms. Thus, we highlight a mechanism in ASCs to sustain the homeostasis of IgG antibody transcripts by integrating Ighg1 mRNA polyadenylation and m6A modification.

EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication.

Efficient DNA replication requires highly coordinated programs for the timely recruitment of protein complexes to DNA replication forks. Defects in this process result in replication stress, which in turn activates cell cycle checkpoints, suppresses cell proliferation and induces apoptosis. In response to persistent cell growth signals that speed up DNA replication processes, cells accelerate the recruitment of DNA replication proteins to avoid DNA replication stress. The mechanisms by which cell growth signals induce processes to facilitate the recruitment of DNA replication proteins onto the replication sites remain unclear. Here, we report that the epidermal growth factor receptor (EGFR) phosphorylates heat shock protein 70 (HSP70) for DNA replication. Such a modification promotes nuclear localization and chromatin association of HSP70, which interacts with the DNA replication coordinator, proliferating cell nuclear antigen (PCNA). HSP70 subsequently facilitates the loading of PCNA onto chromatin. Knockdown or chemical inhibition of HSP70 suppresses PCNA association with chromatin and impairs DNA synthesis and Okazaki fragment maturation, leading to replicative DNA double-strand breaks and apoptosis. Furthermore, chemical inhibition of HSP70 potentiates EGFR-tyrosine kinase inhibitor-induced tumor reduction in vivo. This work expands our understanding of oncogenesis-induced DNA replication processes and provides a foundation for improved treatments for EGFR-mutated lung cancer by simultaneously targeting HSP70.

Dysfunctional atrial fibroblast processing of filamin A in atrial fibrillation

Abstract Background/Introduction Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, is associated with atrial structural remodelling, hallmarked by fibrosis. Atrial fibrosis is instigated by atrial fibroblasts (AFBs). It was previously uncovered the hormone calcitonin (CT) is produced in the heart and, via binding to the CT-receptors (CTR), inhibits atrial fibrogenesis and fibroblast profibrotic activity. [1] In AF, abnormally increased CTR internalisation causes loss of physiological surface CTR and prevents fibroblast activation by CT. The molecular mechanisms of this phenomenon in ACFs are unknown. Aim To explore the role of filamin A (FLNa), an actin cross-linking protein that was previously shown to interact with CTR, [2] in the regulation of CTR trafficking. Methods AFBs were enzymatically isolated from left atrial appendage biopsies collected from 30 patients in persistent AF or normal sinus rhythm (SR, controls) undergoing elective heart surgery. Immunostaining assessed the localisation of CTR and its overlap with the nucleus (stained with DAPI) and FLNa in AFBs. Co-localisation was quantified by the JACoP BIOP plugin for ImageJ. Target mRNA expression was assessed by RT-qPCR and protein levels by immunoblot. Immunoprecipitation (IP) then immunoblot (IB) validated protein-protein interactions. Calpain activity was assessed with a fluorescence-based assay. The effects of calpains on FLNa proteolytic processing was examined with calpain inhibitor calpeptin (10nM–100nM vs DMSO loading control). Results Immunostaining revealed aberrant, intracellular localisation of CTR in AF-AFBs (A) with increased nuclear localisation (p=0.03) and reduced co-localisation with FLNa (p=0.03). FLNa gene expression showed a trend towards reduction in AF (by 36%, p=0.06; B). Meanwhile, although full-length FLNa protein was unaltered in AF, there was a striking 67% reduction (p=0.02) in the expression of FLNa’s c-terminal fragment (FLNaCT), which contains the CTR binding domain (C). Co-immunoprecipitation confirmed a physical interaction between CTR and FLNaCT (D) in both SR and AF AFBs. FLNaCT is typically generated when FLNa is cleaved by the protease calpain 1, whose protein (F) but not mRNA (E), was significantly downregulated (71%, p<0.001) in AF. Puzzlingly, a ~70% loss of calpain 1 protein was associated with unchanged calpain activity (G) and FLNaCT levels in AFBs treated with calpain inhibitor calpeptin (H). Conclusions Persistent AF is associated with an aberrant CTR localisation and FLNa proteolytic processing, not attributable to altered calpain activity. Further characterisation of the molecular determinants of the altered CTR localisation may uncover novel and potentially druggable facets of structural remodelling in AF.

Investigating the role of an orphan nuclear receptor NR4A1 in atrial fibrillation

Abstract Introduction Atrial fibrosis is a major remodelling process in atrial fibrillation (AF). The lack of clinically effective drug targets, due to insufficient understanding of the mechanisms of cardiac fibrosis, hampers the treatment of AF. Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is linked to fibrotic disease in multiple organs, however, its role in cardiac fibrosis and AF is yet to be demonstrated. Recent transcriptomic profiling revealed that NR4A1 expression is downregulated in human atrial cardiac fibroblasts (ACFs) in the presence of persistent AF. However, its mechanistic role in atrial fibrogenesis and AF is unknown. Purpose To investigate the expression profile and function of NR4A1 in human ACFs and provide insights into the role of NR4A1 in the context of persistent AF. Methods Human ACFs were isolated (enzymatic digestion) from right and left atrial appendages of 24 patients with persistent AF and controls in sinus rhythm (SR), who had elective heart surgery. NR4A1 knockdown in ACFs was achieved using NR4A1-siRNA. Gene and protein expression were determined by qPCR and western blot respectively, while BrdU assay and scratch assay were used to evaluate cell proliferation and migration. Atrial fibroblasts subpopulations were identified by single-nucleus RNA-sequencing. Subcellular localisation of NR4A1 was assessed by immunostaining, analysed by ImageJ. Results Validation of RNA-sequencing data confirmed a reduction of NR4A1 gene expression in human ACFs in persistent AF by 25% (p=0.029, Fig. 1A) and the NR4A1 predominant abundance in one of three identified subclusters of human ACFs (NR4A1+NAMPT+). The siRNA-mediated knockdown of NR4A1 in ACFs suppressed gene and protein expression of important component of extracellular matrix collagen-1 (Fig. 1B and Fig. 1C) and fibronectin protein (but not mRNA). The NR4A1-deficient cells also had decreased alpha-smooth muscle actin (αSMA) protein and gene expression (Fig. 1D and Fig. 1E), reduced periostin gene expression in the absence of changes in its protein. Furthermore, the NR4A1-deficiency reduced proliferation (by 18%, p=0.005, Fig. 1F) and accelerated migration (p=0.019, Fig. 1G) of human ACFs. These effects were independent of TGF-β1 stimulation, unlike reported in rat neonatal cardiac and dermal fibroblasts. Despite decreased NR4A1 gene expression, patients with AF had doubled NR4A1 protein levels compared to SR-ACFs (p=0.049, Fig. 2A). This was associated with abnormal 1.8-fold increase in the receptor subcellular localisation (p=0.032, Fig. 2B and Fig. 2C), as opposed to the physiological nuclear localisation observed in ACFs in the absence of AF. Conclusion While under physiological conditions, NR4A1 potently activates profibrotic processes in human ACFs, persistent AF patients have increased but abnormally distributed NR4A1 protein in atrial fibroblasts, whose consequences are yet to be determined in order to develop new therapies for AF.Figure 1Figure 2

DNA Hypomethylation Activates the RpMYB2-Centred Gene Network to Enhance Regeneration of Adventitious Roots.

Plants, being immobile, are exposed to environmental adversities such as wind, snow and animals that damage their structure, making regeneration essential for their survival. The adventitious roots (ARs) primarily emerge from a detached explant to uptake nutrients; therefore, the molecular network involved in their regeneration needs to be explored. DNA methylation, a key epigenetic mark, influences molecular pathways, and recent studies suggested its role in regeneration. In our research, the application of 5-azacytidine (5-azaC), an inhibitor of DNA methylation, caused the earlier initiation and development of root primordia and consequently enhanced the AR regeneration rate in Robinia psuedoacacia L (black locust). The whole-genome bisulfite sequencing (WGBS) revealed a decrease in global methylation and an increase in hypomethylated cytosine sites and regions across all contexts including CHH, CHG and mergedCG caused transcriptional variations in 5-azaC-treated sample. The yeast two-hybrid (Y2H) assay revealed a RpMYB2-centred network of transcriptionally activated transcription factors (TFs) including RpWRKY23, RpGATA23, RpSPL16 and other genes like RpSDP, RpSS1, RpBEN1, RpGULL05 and RpCUV with nuclear localization suggesting their potential co-localization. Additionally, yeast one-hybrid (Y1H) assay showed the interaction of RpMYB2 interactors, RpGATA23 and RpWRKY23, with promoters of RpSK6 and RpCDC48, and luciferase reporting assay (LRA) validated their binding with RpSK6. Our results revealed that hypomethylation-mediated transcriptomic modifications activated the RpMYB2-centred gene network to enhance AR regeneration in black locust hypocotyl cuttings. These findings pave the way for genetic modification to improve plant regeneration ability and increase wood production while withstanding environmental damage.

METTL3 drives heart failure by regulating Spp1 and Fos m6A modification in myocardial infarction

Abstract Background While m6A modification has been reported in myocardial infarction (MI), the detailed mechanism by which METTL3 regulates the progression of the disease has not yet been elucidated, and it remains unclear why m6A modification increases after MI. Purpose To investigate the role and mechanism of METTL3 in regulating its targets through m6A methylation in MI. The research results will not only add new content to the basic mechanism of myocardial protection but also provide new ideas and new targets for the prevention and treatment of MI. Methods Through MeRIP-seq and extensive bioinformatics analysis, the target genes SPP1 and FOS with the most significant m6A modification and differential expression in MI were screened. We successfully constructed heart specific Mettl3 knockout mice (Mettl3CKO) to verify that METTL3 promotes the deterioration of cardiac function after MI. We performed complementary molecular methods to assess protein quantity and interactions to identify mechanisms regulating this response. We manipulated select molecular pathways using both genetic and pharmacological methods to validate these mechanisms. Results Here, we showed that METTL3 exerted methyltransferase activity-dependent functions in gene regulation in MI, and a significant transcription factor HuR assisted the function of METTL3. and demonstrated that METTL3 was critical for the promotion of heart failure after MI. More specifically, METTL3 directly interacted with HuR through its nuclear localization domain in the cell nucleus under normoxia condition. When hypoxia developed, METTL3 separated from HuR and deposited m6A into 5’UTR of Spp1 and Fos mRNA to maintain their stability. In contrast, HuR bound to the ARE domain of 3’UTR of Spp1 and Fos mRNA to take them to the cytosol, maintaining their stability. Moreover, HIF-1α directly interacted with the HRE domain of Mettl3 to promote its transcription, and HuR bound to the ARE domain of 3’UTR of Mettl3 mRNA to maintain its stability to promote following translation. Conclusions Collectively, our studies revealed previously unappreciated functions of METTL3 with the help of HuR, and a direct target of HIF-1α under normoxia condition, which together contribute to its essential function in MI, suggesting therapeutic potential for targeting the METTL3/HuR/Spp1(Fos) mRNA axis.Figure1

Adsorption and Distribution of Triplex-Hybridizing Bioconjugated Gold Nanoclusters Inside Spermatozoa- A Study Using Confocal Microscopy and Fluorescence Lifetime Imaging.

The study of the interactions between biofunctionalized gold nanoclusters (Au NCs) and spermatozoa is highly relevant to evaluate the potential of Au NCs as imaging probes and transfection agents in reproductive biology. In this work, confocal laser scanning microscopy (CLSM) was used to investigate the distribution of Au NCs bioconjugated with peptide (nuclear localisation sequence, NLS) and oligonucleotide (locked nucleic acid, LNA) ligands in bovine spermatozoa. Fluorescence lifetime imaging (FLIM) was employed to detect changes in the NC's chemical environment. We observed a pronounced regio-selective accumulation of the bioconjugates in spermatozoa with high concentration at the equatorial segment. Furthermore, 3D-CLSM showed successful non-endosomal cellular uptake of the conjugates by intact sperm cells and the distribution of the bioconjugates was found to be influenced by the ligand types. Interestingly, the FLIM data showed differences in lifetime depending on membrane integrity. Furthermore, ligand-dependent changes in lifetime between NC bioconjugates carrying peptide and oligonucleotide ligands were found, probably attributed to specific interactions with sperm cell compartments.

Characterization of Hippo Signaling Components in the Early Dorsal Pancreatic Bud.

YAP1 expression in the early mouse pancreatic anlagen is low until approximately E11.5, when it becomes localized to cell nuclei in multipotent progenitor cells. At E14.5, we find nuclear YAP1 in ductal cells.YAP1 is not expressed in early and midgestation endocrine cells. By contrast, TAZ is expressed in first transition endocrine cells.Hippo regulators MERLIN and LATS1/2 kinases are robustly expressed in the early pancreatic bud by E10.5. Both MERLIN and LATS1/2 exhibit strong apical localization in epithelial cells at nascent microlumens. Using in vitro models of de novo pancreas lumen formation, we show that YAP1 nuclear localization is high in early phases of lumen formation and gradually decreases as lumens matures.

Chemomechanical regulation of EZH2 localization controls epithelial-mesenchymal transition.

The methyltransferase enhancer of zeste homolog 2 (EZH2) regulates gene expression and aberrant EZH2 expression and signaling can drive fibrosis and cancer. However, it is not clear how chemical and mechanical signals are integrated to regulate EZH2 and gene expression. We show that culture of cells on stiff matrices in concert with transforming growth factor (TGF)-β1 promotes nuclear localization of EZH2 and an increase in the levels of the corresponding histone modification, H3K27me3, thereby regulating gene expression. EZH2 activity and expression are required for TGFβ1- and stiffness-induced increases in H3K27me3 levels as well as for morphological and gene expression changes associated with epithelial-mesenchymal transition (EMT). Inhibition of Rho associated kinase (ROCK) or myosin II signaling attenuates TGFβ1-induced nuclear localization of EZH2 and decreases H3K27me3 levels in cells cultured on stiff substrata, suggesting that cellular contractility, in concert with a major cancer signaling regulator TGFβ1, modulates EZH2 subcellular localization. These findings provide a contractility-dependent mechanism by which matrix stiffness and TGFβ1 together mediate EZH2 signaling to promote EMT.