Protein Nuclear Localization Research Articles

Divergent Effects of Circoviridae Capsid Proteins on Type I Interferon Signaling

Viruses in the Circoviridae family can infect mammals and birds. Porcine circovirus type 2 (PCV2) significantly affects the livestock industry by causing porcine circovirus-associated diseases, such as postweaning multisystem wasting syndrome, respiratory disease complex, and dermatitis nephropathy syndrome. Additionally, beak and feather disease virus in parrots, canine circovirus in dogs, and columbid circovirus (pigeon circovirus) in racing pigeons induce immunosuppression, followed by secondary infections in these hosts. Although the PCV2 capsid protein has been demonstrated to inhibit type I interferon (IFN) signaling, the molecular mechanisms of Circoviridae-induced immunosuppression are largely unknown. In this study, we examined whether these functions are conserved across Circoviridae capsid proteins. Our results illustrated that although the nuclear localization of capsid proteins is conserved, their effects on IFN-β signaling vary by species, revealing the diverse roles of Circoviridae capsid proteins in modulating immune responses.

Click hydrogels to assess stiffness-induced activation of pancreatic cancer-associated fibroblasts and its impact on cancer cell spreading.

Pancreatic ductal adenocarcinoma (PDAC) is marked by significant desmoplastic reactions, or the accumulation of excessive extracellular matrices. PDAC stroma has abnormally high stiffness, which alters cancer cell behaviors and creates a barrier for effective drug delivery. Unfortunately, clinical trials using a combination of chemotherapy and matrix-degrading enzyme have led to disappointing results, as the degradation of stromal tissue likely accelerated the dissemination of cancer cells. High matrix stiffness has been shown to activate cancer-associated fibroblasts (CAFs), increasing their interaction with pancreatic cancer cells (PCCs) through promoting proliferation, migration, and resistance to chemotherapy. With the advance of biomaterials science and engineering, it is now possible to design chemically defined matrices to understand the role of stiffness in activating pancreatic CAFs and how this may alter cancer cell migration. Here, we developed a norbornene-based click hydrogel system with independently tunable stiffness and cell adhesive ligand to evaluate stiffness-induced activation of CAFs and migration of PCCs. Our results show that matrix stiffness did not alter matrix deposition from CAFs but affected nuclear localization of Yes-associated protein (YAP). Our results also verify the role of CAFs on promoting PCC migration and an elevated substrate stiffness further increased PCC motility.

Retinoic Acid Improves Vascular Endothelial Dysfunction by Inhibiting PI3K/AKT/YAP-Mediated Ferroptosis in Diabetes Mellitus.

Vascular endothelial dysfunction is the initial factor involved in cardiovascular injury in patients with diabetes. Retinoic acid is involved in improving vascular complications in patients with diabetes, but its protective mechanism is still unclear. This study aimed to evaluate the effect and mechanism of All-Trans Retinoic Acid (ATRA) on endothelial dysfunction induced by diabetes. In the present study, streptozotocin (STZ)-induced diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were observed, and the effects of ATRA on HG-induced endothelial dysfunction and ferroptosis were evaluated. ATRA treatment improved impaired vasorelaxation in diabetic aortas in an endothelium-dependent manner, and this effect was accompanied by an increase in the NO concentration and eNOS expression. Ferroptosis, characterized by lipid peroxidation and iron overload induced by HG, was improved by ATRA administration, and a ferroptosis inhibitor (ferrostatin-1, Fer-1) improved endothelial function to a similar extent as ATRA. In addition, the inactivation of phosphoinositol-3-kinase (PI3K)/protein kinases B (AKT) and Yes-Associated Protein (YAP) nuclear localization induced by HG were reversed by ATRA administration. Vascular ring relaxation experiments showed that PI3K/AKT activation and YAP inhibition had similar effects on ferroptosis and endothelial function. However, the vasodilative effect of retinoic acid was affected by PI3K/AKT inhibition, and the inhibitory effects of ATRA on ferroptosis and the improvement of endothelial function were dependent on the retinoic acid receptor. ATRA could improve vascular endothelial dysfunction by inhibiting PI3K/AKT/YAP-mediated ferroptosis induced by HG, which provides a new idea for the treatment of vascular lesions in diabetes.

Importin α inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture. As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites). Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested. Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation. As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs.

Follistatin From hiPSC-Cardiomyocytes Promotes Myocyte Proliferation in Pigs With Postinfarction LV Remodeling.

When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes (CCND2-OEhiPSC-CMs) and administered to the infarcted hearts of immunodeficient mice, the cells proliferated after administration and repopulated >50% of the scar. Here, we knocked out human leukocyte antigen class I and class II expression in CCND2-OEhiPSC-CMs (KO/OEhiPSC-CMs) to reduce the cells' immunogenicity and then assessed the therapeutic efficacy of KO/OEhiPSC-CMs for the treatment of myocardial infarction. KO/OEhiPSC-CM and wild-type hiPSC-CM (WThiPSC-CM) spheroids were differentiated in shaking flasks, purified, characterized, and intramyocardially injected into pigs after ischemia/reperfusion injury; control animals were injected with basal medium. Cardiac function was evaluated via cardiac magnetic resonance imaging, and cardiomyocyte proliferation was assessed via immunostaining and single-nucleus RNA sequencing. Measurements of cardiac function and scar size were significantly better in pigs treated with KO/OEhiPSC-CM spheroids than in animals treated with medium or WThiPSC-CM spheroids. KO/OEhiPSC-CMs were detected for just 1 week after administration, but assessments of cell cycle activity and proliferation were significantly higher in the endogenous pig cardiomyocytes of the hearts from the KO/OEhiPSC-CM spheroid group than in those from the other 2 groups. Single-nucleus RNA-sequencing analysis identified a cluster of proliferating cardiomyocytes that was significantly more prevalent in the KO/OEhiPSC-CM spheroid-treated hearts (3.65%) than in the hearts from the medium (0.89%) or WThiPSC-CM spheroid (1.33%) groups at week 1. YAP (Yes-associated protein) protein levels and nuclear localization were also significantly upregulated in pig cardiomyocytes after treatment with KO/OEhiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from KO/OEhiPSC-CM spheroids than WThiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, P=0.0056). Treatment with follistatin increased WThiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice. KO/OEhiPSC-CM spheroids significantly improved cardiac function and reduced infarct size in pig hearts after ischemia/reperfusion injury by secreting follistatin, which upregulated HIPPO/YAP signaling and proliferation in endogenous pig cardiomyocytes.

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

Dual roles of pear EARLY FLOWERING 4 -like genes in regulating flowering and leaf senescence

BackgroundFlowering is a critical agronomic trait in fruit tree cultivation, essential for sexual reproduction and fruit yield. Circadian clock system, governing processes such as flowering, growth, and hormone signaling, plays a key role in plant adaptability. While some clock-related genes influencing pear flowering have been studied, the role of the PbELF4 (EARLY FLOWERING 4) family remains largely unexplored.ResultsIn this study, we identified five ELF4 homologous genes within the pear (Pyrus bretschneideri) genome. Phylogenetic analysis delineated two distinct groups within the PbELF4 genes, with PbELF4a and PbELF4b clustering with AtELF4. Expression profiling across various pear tissues revealed diverse expression patterns. Diurnal rhythms of PbELF4 genes were discernible in pear leaves, suggesting potential regulatory roles. Ectopic overexpression of PbELF4a and PbELF4b in Arabidopsis significantly delayed flowering and suppressed the expression of flowering-related genes. Additionally, PbELF4b overexpression induced premature leaf senescence, evidenced by reduced chlorophyll content and increased expression of senescence-associated genes. Nuclear localization of PbELF4a and PbELF4b proteins was observed, and interaction assays revealed that PbELF4a interacted with PbELF3α.ConclusionsThese findings underscore the conserved function of PbELF4a and PbELF4b as negative regulators of flowering time, with PbELF4b also demonstrating a positive role in leaf senescence.

Genome-wide identification and analysis of ERF transcription factors related to abiotic stress responses in Nelumbo nucifera

BackgroundEthylene-responsive factor (ERF) transcription factors belong to the APETALA2/ERF (AP2/ERF) superfamily, and play crucial roles in plant development process and stress responses. However, the function of ERF proteins (especially for their role in response to abiotic stresses) remains scarce in Nelumbo nucifera, which is an important aquatic plant with high ornamental, economic, and ecological values.ResultsA total of 107 ERF genes were identified from the N. nucifera genome, and phylogenetic analysis classified these genes into 11 groups. The NnERF genes in the same group exhibited similar gene structure and conserved motifs, and they were unevenly distributed across the 8 chromosomes, with three pairs of tandem duplications and 21 pairs of segmental duplications. Synteny analysis revealed 44 and 39 of NnERF genes were orthologous to those in Arabidopsis thaliana and Oryza sativa, respectively. Tissue-specific expression patterns analysis of NnERF showed that 26 NnERF genes were expressed in all tested tissues, in which five genes exhibited high expression levels. Furthermore, 16 NnERF genes were selected for exploring their responses to different abiotic stresses, including cold, salt, drought, and Cd stresses. qRT-PCR analysis revealed that all these 16 investigated genes were regulated by at least one stress treatment, and 12 genes responded to all the stress treatments with different expression patterns or levels, suggesting their potential roles in diverse abiotic stress tolerance of N. nucifera. Additionally, two representative stress-related NnERFs (Nn3g19628 and Nn1g06033) were confirmed to be nuclear-localized proteins and displayed transcriptional activation.ConclusionsIn this study, we conducted a genome-wide identification and analysis of NnERF gene family related to abiotic stress responses in N. nucifera, which provides valuable information for further functional validation of these genes in stress responses, and forms a foundation for stress tolerance breeding in N. nucifera and other aquatic ornamental plants.

The SBP-box transcription factor PlSPL2 negatively regulates stem development in herbaceous peony.

The SBP-box transcription factor PlSPL2 silencing in herbaceous peony enhanced stem strength by regulating xylem development, whereas its overexpression in tobacco resulted in weaker stem strength and undeveloped xylem. The strength of plant stems is a critical determinant of lodging resistance of plants, which significantly affects crop yield and cut-flower quality. Squamosa promoter binding (SBP) protein-like (SPL) transcription factors (TFs), participate in multiple regulatory processes, particularly in stem development. In this study, PlSPL2, an orthologous gene of Arabidopsis AtSPL2 in herbaceous peony, was isolated and found to contain a conserved SBP domain featuring two typical Zn-binding sites, as well as a nuclear localization sequence (NLS). Subsequently, transient infection of tobacco leaf epidermal cells using Agrobacterium confirmed the nuclear localization of PISPL2 protein. Additionally, gene expression analyses revealed that PlSPL2 was preferentially expressed in stems, and demonstrated a download trend in expression levels within vascular bundles during stem cell wall development. Furthermore, silencing of PlSPL2 in herbaceous peony enhanced stem strength. The silenced plants exhibited more developed xylems with wider radii and higher numbers of cell layers. Overexpression of PlSPL2 in tobacco, however, resulted in weaker stem strength, accompanied by a narrower radius of the xylem. These findings suggested that PlSPL2 was a negative regulator of herbaceous peony stem development, and its discovery and research could significantly contribute to a deeper understanding of stem growth and development mechanisms.

Identification and Characterization of EIN3/EIL Transcription Factor Family Members in Pinus massoniana Lamb.

Transcription factors refer to types of proteins that perform significant functions in the process of gene expression regulation. The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) family, functioning as significant transcription factors regulating ethylene, plays a critical role in the growth and development of plants and participates in the plant's response to diverse environmental stresses. Pinus massoniana is an excellent native tree with high economic and ecological value. However, the study of EIN3/EIL genes in gymnosperms, for instance, P. massoniana, is still relatively limited. In this research, four putative EIN3/EIL genes were identified in the transcriptome of P. massoniana. Bioinformatics analysis showed that PmEIL genes contain a highly conserved EIN3 domain and other structural features of acidic, proline-rich and glutamine-rich sites. The molecular evolution tree analysis demonstrated that the EIN3/EIL family was partitioned into three categories (A, B, and C), and the number, type, and distribution of conserved motifs grouped in one category were similar. The results of qRT-PCR indicated that the expression levels of PmEIL genes were markedly elevated in needles compared to other tissues. Through the analysis of expression patterns of the PmEIL genes under various stress treatments, it was found that the PmEIL genes could participate in plant hormone stimulation induction, osmosis, drought and other response processes. In addition, PmEIL is a nuclear localization protein. PmEIL1, PmEIL3, and PmEIL4 are transcriptional activators, while PmEIL2 is a transcriptional suppressor. This research provides a basis for further elucidating the function of EIN3/EIL transcription factors in growth, development and stress response of P. massoniana.

LncRNA Snhg3 aggravates hepatic steatosis via PPARγ signaling.

LncRNAs are involved in modulating the individual risk and the severity of progression in metabolic dysfunction-associated fatty liver disease (MASLD), but their precise roles remain largely unknown. This study aimed to investigate the role of lncRNA Snhg3 in the development and progression of MASLD, along with the underlying mechanisms. The result showed that Snhg3 was significantly downregulated in the liver of high-fat diet-induced obesity (DIO) mice. Notably, palmitic acid promoted the expression of Snhg3 and overexpression of Snhg3 increased lipid accumulation in primary hepatocytes. Furthermore, hepatocyte-specific Snhg3 deficiency decreased body and liver weight, alleviated hepatic steatosis and promoted hepatic fatty acid metabolism in DIO mice, whereas overexpression induced the opposite effect. Mechanistically, Snhg3 promoted the expression, stability and nuclear localization of SND1 protein via interacting with SND1, thereby inducing K63-linked ubiquitination modification of SND1. Moreover, Snhg3 decreased the H3K27me3 level and induced SND1-mediated chromatin loose remodeling, thus reducing H3K27me3 enrichment at the Pparg promoter and enhancing PPARγ expression. The administration of PPARγ antagonist T0070907 improved Snhg3-aggravated hepatic steatosis. Our study revealed a new signaling pathway, Snhg3/SND1/H3K27me3/PPARγ, responsible for mice MASLD and indicates that lncRNA-mediated epigenetic modification has a crucial role in the pathology of MASLD.

LncRNA Snhg3 aggravates hepatic steatosis via PPARγ signaling

LncRNAs are involved in modulating the individual risk and the severity of progression in metabolic dysfunction-associated fatty liver disease (MASLD), but their precise roles remain largely unknown. This study aimed to investigate the role of lncRNA Snhg3 in the development and progression of MASLD, along with the underlying mechanisms. The result showed that Snhg3 was significantly downregulated in the liver of high-fat diet-induced obesity (DIO) mice. Notably, palmitic acid promoted the expression of Snhg3 and overexpression of Snhg3 increased lipid accumulation in primary hepatocytes. Furthermore, hepatocyte-specific Snhg3 deficiency decreased body and liver weight, alleviated hepatic steatosis and promoted hepatic fatty acid metabolism in DIO mice, whereas overexpression induced the opposite effect. Mechanistically, Snhg3 promoted the expression, stability and nuclear localization of SND1 protein via interacting with SND1, thereby inducing K63-linked ubiquitination modification of SND1. Moreover, Snhg3 decreased the H3K27me3 level and induced SND1-mediated chromatin loose remodeling, thus reducing H3K27me3 enrichment at the Pparg promoter and enhancing PPARγ expression. The administration of PPARγ antagonist T0070907 improved Snhg3-aggravated hepatic steatosis. Our study revealed a new signaling pathway, Snhg3/SND1/H3K27me3/PPARγ, responsible for mice MASLD and indicates that lncRNA-mediated epigenetic modification has a crucial role in the pathology of MASLD.

A High-Throughput Screen for Antiproliferative Peptides in Mammalian Cells Identifies Key Transcription Factor Families.

Transcription factors (TFs) are a promising therapeutic target for a multitude of diseases. TFs perform their cellular roles by participating in multiple specific protein-protein interactions. For example, homo- or heterodimerization of some TFs controls DNA binding, while interactions between TFs and components of basal transcriptional machinery or chromatin modifiers can also be critical. While, in theory, small molecules could be used to disrupt specific protein-protein interfaces required for TF function, in practice, it is difficult to identify small molecules with the necessary specificity and efficacy, likely due to the extensive protein-protein interfaces that often underlie TF function. However, in contrast to small molecules, peptides have the potential to provide both the specificity and efficacy required to disrupt such interfaces. Here, we identified ∼15 peptides that inhibit the proliferation of leukemia cells using a high-throughput pooled screen of a library of 80-mer protein regions (peptides) derived from human nuclear-localized proteins. The antiproliferative peptides were enriched for regions known to be involved in specific TF dimerization, including the basic leucine zipper (bZIP) domain family. One of these bZIP domains, JDP2;bZIP_1, from the TF JDP2, was the top antiproliferative peptide, reducing the proliferation of K562 cells by 2-fold. JDP2;bZIP_1 inhibited AP-1 transcriptional activity and phenocopied JDP2 overexpression, suggesting that the peptide affected proliferation through a native JDP2 mechanism. Unexpectedly, given the strong conservation of the bZIP domain, residues outside of the annotated dimerization domain were critical for the peptide's antiproliferative potency. The peptide-mediated antiproliferative effect initiated erythrocyte differentiation in K562 cells and increased G0/G1 cells across multiple cell line models. We also found that many of the antiproliferative peptides identified in this study, including JDP2;bZIP_1, did not require a nuclear localization signal to function, a potential benefit for delivering these peptides in therapeutic applications.

HSPA12A stimulates "Smurf1-Hif1α-aerobic glycolysis" axis to promote proliferation of renal tubular epithelial cells after hypoxia/reoxygenation injury.

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12h) and reoxygenation (12h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacologicalinhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R.

Identification of a Novel Gene MtbZIP60 as a Negative Regulator of Leaf Senescence through Transcriptome Analysis in Medicago truncatula.

Leaves are the primary harvest portion in forage crops such as alfalfa (Medicago sativa). Delaying leaf senescence is an effective strategy to improve forage biomass production and quality. In this study, we employed transcriptome sequencing to analyze the transcriptional changes and identify key senescence-associated genes under age-dependent leaf senescence in Medicago truncatula, a legume forage model plant. Through comparing the obtained expression data at different time points, we obtained 1057 differentially expressed genes, with 108 consistently up-regulated genes across leaf growth and senescence. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the 108 SAGs mainly related to protein processing, nitrogen metabolism, amino acid metabolism, RNA degradation and plant hormone signal transduction. Among the 108 SAGs, seven transcription factors were identified in which a novel bZIP transcription factor MtbZIP60 was proved to inhibit leaf senescence. MtbZIP60 encodes a nuclear-localized protein and possesses transactivation activity. Further study demonstrated MtbZIP60 could associate with MtWRKY40, both of which exhibited an up-regulated expression pattern during leaf senescence, indicating their crucial roles in the regulation of leaf senescence. Our findings help elucidate the molecular mechanisms of leaf senescence in M. truncatula and provide candidates for the genetic improvement of forage crops, with a focus on regulating leaf senescence.

Expansion in situ genome sequencing links nuclear abnormalities to hotspots of aberrant euchromatin repression.

Microscopy and genomics are both used to characterize cell function, but approaches to connect the two types of information are lacking, particularly at subnuclear resolution. While emerging multiplexed imaging methods can simultaneously localize genomic regions and nuclear proteins, their ability to accurately measure DNA-protein interactions is constrained by the diffraction limit of optical microscopy. Here, we describe expansion in situ genome sequencing (ExIGS), a technology that enables sequencing of genomic DNA and superresolution localization of nuclear proteins in single cells. We applied ExIGS to fibroblast cells derived from an individual with Hutchinson-Gilford progeria syndrome to characterize how variation in nuclear morphology affects spatial chromatin organization. Using this data, we discovered that lamin abnormalities are linked to hotspots of aberrant euchromatin repression that may erode cell identity. Further, we show that lamin abnormalities heterogeneously increase the repressive environment of the nucleus in tissues and aged cells. These results demonstrate that ExIGS may serve as a generalizable platform for connecting nuclear abnormalities to changes in gene regulation across disease contexts.

Endosperm-specific expressed transcription factor protein WRINKLED1-mediated oil accumulative mechanism in woody oil peony Paeonia ostii var. lishizhenii

Paeonia ostii var. lishizhenii exhibits superiority of high α-linolenic acid in seed oils, yet, the low yield highlights the importance of enhancing oil accumulation in seeds for edible oil production. The transcription factor protein WRINKLED1 (WRI1) plays crucial roles in modulating oil content in higher plants; however, its functional characterization remains elusive in P. ostii var. lishizhenii. Herein, based on a correlation analysis of transcription factor transcript levels, FA accumulation rates, and interaction assay of FA biosynthesis associated proteins, a WRI1 homologous gene (PoWRI1) that potentially regulated oil content in P. ostii var. lishizhenii seeds was screened. The PoWRI1 exhibited an endosperm-specific and development-depended expression pattern, encoding a nuclear-localized protein with transcriptional activation capability. Notably, overexpressing PoWRI1 upregulated certain key genes relevant to glycolysis, FA biosynthesis and desaturation, and improved seed development, oil body formation and oil accumulation in Arabidopsis seeds, resulting an enhancement of total seed oil weight by 9.47–18.77 %. The defective impacts on seed phenotypes were rescued through ectopic induction of PoWRI1 in wri1 mutants. Our findings highlight the pivotal role of PoWRI1 in controlling oil accumulation in P. ostii var. lishizhenii, offering bioengineering strategies to increase seed oil accumulation and enhance its potential for edible oil production.

FANS Unfixed: Isolation and Proteomic Analysis of Mouse Cell Type-Specific Brain Nuclei.

Epigenetic-mediated gene regulation orchestrates brain cell-type gene expression programs, and epigenetic dysregulation is a major driver of aging and disease-associated changes. Proteins that mediate gene regulation are mostly localized to the nucleus; however, nuclear-localized proteins are often underrepresented in gene expression studies and have been understudied in the context of the brain. To address this challenge, we have optimized an approach for nuclei isolation that is compatible with proteomic analysis. This was coupled to a mass spectrometry protocol for detecting proteins in low-concentration samples. We have generated nuclear proteomes for neurons, microglia, and oligodendrocytes from the mouse brain cortex and identified cell-type nuclear proteins associated with chromatin structure and organization, chromatin modifiers such as transcription factors, and RNA-binding proteins, among others. Our nuclear proteomics platform paves the way for assessing brain cell type changes in the nuclear proteome across health and disease, such as neurodevelopmental, aging, neurodegenerative, and neuroinflammatory conditions. Data are available via ProteomeXchange with the identifier PXD053515.

Genome-wide identification of R2R3-MYB transcription factors in Betula platyphylla and functional analysis of BpMYB95 in salt tolerance

The Myeloblastosis (MYB) transcription factor (TF) family is one of the largest transcription factor families in plants and plays an important role in various physiological processes. At present, there are few reports on birch (Betula platyphylla Suk.) of R2R3-MYB-TFs, and most BpMYBs still need to be characterized. In this study, 111 R2R3-MYB-TFs with conserved R2 and R3 MYB domains were identified. Phylogenetic tree analysis showed that the MYB family members of Arabidopsis thaliana and birch were divided into 23 and 21 subgroups, respectively. The latter exhibited an uneven distribution across 14 chromosomes. There were five tandem duplication events and 17 segmental duplication events between BpMYBs, and repeat events play an important role in the expansion of the family. In addition, the promoter region of MYBs was rich in various cis-acting elements, and MYB-TFs were involved in plant growth and development, light responses, biotic stress, and abiotic stress. RNA-sequencing (RNA-seq) and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results revealed that most R2R3-MYB-TFs in birch responded to salt stress. In particular, the expression of BpMYBs in the S20 subfamily was significantly induced by salt, drought, abscisic acid, and methyl jasmonate stresses. Based on the weighted co-expression network analysis of physiological and RNA-seq data of birch under salt stress, a key MYB-TF BpMYB95 (BPChr12G24087), was identified in response to salt stress, and its expression level was induced by salt stress. BpMYB95 is a nuclear localization protein with transcriptional activation activity in yeast and overexpression of this gene significantly enhanced salt tolerance in Saccharomyces cerevisiae. The qRT-PCR and histochemical staining results showed that BpMYB95 exhibited the highest expression in the roots, young leaves, and petioles of birch plants. Overexpression of BpMYB95 significantly improved salt-induced browning and wilting symptoms in birch leaves and alleviated the degree of PSII photoinhibition caused by salt stress in birch seedlings. In conclusion, most R2R3-MYB-TFs found in birch were involved in the salt stress response mechanisms. Among these, BpMYB95 was a key regulatory factor that significantly enhanced salt tolerance in birch. The findings of this study provide valuable genetic resources for the development of salt-tolerant birch varieties.

AT-hook motif nuclear localized transcription factors function redundantly in promoting root growth through modulation of redox homeostasis.

Maintaining an optimal redox status is essential for plant growth and development, particularly when the plants are under stress. AT-hook motif nuclear localized (AHL) proteins are evolutionarily conserved transcription factors in plants. Much of our understanding about this gene family has been derived from studies on clade A members. To elucidate the functions of clade B genes, we first analyzed their spatial expression patterns using transgenic plants expressing a nuclear localized GFP under the control of their promoter sequences. AHL1, 2, 6, 7, and 10 were further functionally characterized owing to their high expression in the root apical meristem. Through mutant analyses and transgenic studies, we showed that these genes have the ability to promote root growth. Using yeast one-hybrid and dual luciferase assays, we demonstrated that AHL1, 2, 6, 7, and 10 are transcription regulators and this activity is required for their roles in root growth. Although mutants for these genes did not showed obvious defects in root growth, transgenic plants expressing their fusion proteins with the SRDX repressor motif exhibited a short-root phenotype. Through transcriptome analysis, histochemical staining and molecular genetics experiments, we found that AHL10 maintains redox homeostasis via direct regulation of glutathione transferase (GST) genes. When the transcript level of GSTF2, a top-ranked target of AHL10, was reduced by RNAi, the short-root phenotype in the AHL10-SRDX expressing plant was largely rescued. These results together suggest that AHL genes function redundantly in promoting root growth through direct regulation of redox homeostasis.