Project description:To determine the global gene occupancy by Wiskott - Aldrich syndrome Protein (WASP) we perform ChIP-seq assay in two lymphoblastoid cell lines. We identify WASP-enriched genes, including several WASP-interaction genes previously reported; in addition, our results suggest the implication of WASP in diverse cellular process
Project description:Nitrogen is a crucial nutrient element for plant growth and productivity, with both excess and deficiency in nitrogen fertilizer application posing adverse effects on plants and the environment. The internal mechanisms by which the medicinal plant Epimedium pubescens (E. pubescens) adapts to varying nitrogen levels remain unclear. This study employed one-year-old E. pubescens as the experimental material to systematically analyze the changes in plant growth traits, carbon metabolites, and Icariin-Flavonoids content under different exogenous nitrogen levels. Furthermore, it examined the transcriptional changes in gene expression within E. pubescens in response to varying nitrogen levels. The results showed that under moderate nitrogen levels (7.5 mmol/L NO3-), E. pubescens exhibited increased biomass accumulation and flavonoid synthesis. However, deficient or excessive nitrogen levels (0, 22.5 mmol/L NO3-) significantly inhibited photosynthesis in E. pubescens, reducing the content of starch, soluble sugars, and Icariin-Flavonoids, leading to decreased biomass and accompanied by changes in leaf color (pale green or browning). Transcriptome analysis revealed the underlying molecular mechanisms of these changes in plants regulated by different nitrogen levels. Nitrogen deficiency and excess triggered distinct transcriptional response patterns, with the number of differentially expressed genes (DEGs) peaking at S2 (36 days) under nitrogen deficiency and significantly declining at S3 (48 days), while the number of DEGs under excess nitrogen continuously increased over time from S1 to S3 (12-48 days). Both conditions significantly affected the expression of genes related to carbon and nitrogen metabolism, flavonoid synthesis, and stress response. Based on the correlation analysis of expression levels of genes related to these pathways, growth traits, and metabolite content indicators, we constructed regulatory network diagrams for carbon-nitrogen metabolism and Icariin-flavonoid metabolism-related genes. Furthermore, we identified hub genes that may be involved in regulating Icariin-flavonoid metabolism in response to nitrogen levels, such as UGT (Ebr06G044290), EpF3H (Ebr04G062950), EpCHS5 (Ebr03G073940) (Fig. 9A), UGT (Ebr06G044660), and EpUGT13_A (Ebr06G044210). Additionally, among the DEGs obtained at the S3 stage, we discovered the transcription factors MYB1_CROXC (Ebr04G001770) and MYB12_ARATH (Ebr01G065030). We constructed their associated gene networks under different nitrogen levels, which may primarily regulate the expression of genes like UGT, CHS, and F3H involved in the flavonoid synthesis stage of E. pubescens under varying nitrogen conditions. In summary, this study has revealed the growth performance and the variation patterns of Icariin-Flavonoid metabolism in E. pubescens in response to different exogenous nitrogen levels, as well as their complex underlying mechanisms. Additionally, key genes involved in regulating the synthesis of flavonol glycosides in E. pubescens by nitrogen levels have been identified. This provides an important basis for a deeper understanding of the mechanisms of nitrogen regulation on the growth and secondary metabolism of medicinal plants. Furthermore, it offers theoretical support and potential genetic resources for the efficient utilization of nitrogen fertilizers in E. pubescens cultivation and the breeding of high-nitrogen-use-efficiency varieties.
Project description:To investigate a role of nuclear WASp in T cell development we performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in thymocytes and spleen CD4+ T cells. To pre-process raw ChIP-Seq data, the total number of reads were normalized and aligned against the mouse genome. WASp was enriched at transcription start sites of a large number of protein-coding genes. Many of the WASp-enriched genes were associated with RNA Polymerase II-enriched genes and active epigenetic marks of transcription; H3K4m3, H3K9a, H3K27a, and with the epigenetic mark for active enhancers H3K4m1. To study the distribution of overactive WASpI296T in the thymocyte genome and to identify regions enriched in WASpI296T binding, we performed second round of ChIP-Seq analysis using the WASp F-8 antibody. To detect differences in gene enrichment between thymocytes expressing wildtype WASp or WASpI296T, we applied stringent conditions and subtracted common genes between the two samples. Using this approach, we identify 70 WASpI296T-enriched genes. Functional clustering of these genes revealed that WASpI296T was associated with RNA Polymerase II genes in 11 functional groups of genes.thymocytes and spleen CD4+ T cells. WASp was enriched at transcription start sites of a large number of protein-coding genes.
Project description:The genus Bothrops is responsible for most part of envenomation accidents in Brazil. Bothrops pubescens is an endemic and neglected species in the Brazilian Pampa Biome. The characterization of its venom is essential since there is no data about it and can be helpful in the discovery of active biomolecules and for a better understanding of its action. We used top-down (TDP), native top-down, and bottom-up proteomic (BUP) approaches to characterize the venom of B. pubescens. We were able to identify 89 protein groups with the BUP approach and 40 unique proteoforms with the TDP approach, demonstrating the similarities and peculiarities of B. pubescens venom. We also identified a dimeric L-amino acid oxidase with using native TDP. Here we present for the first time a bothropic venom characterization through TDP approaches.
Project description:Mutations in the immune-specific actin regulator WASp induce a proinflammatory state in myeloid cells, whose underlying causes remain poorly defined. Here, we applied microfabricated tools that mimic tissue mechanical forces to explore the role of WASp in connecting mechano-sensing to the activation of inflammatory responses in macrophages. We show that WASp-deficient macrophages carry nuclear structure alterations and undergo increased blebbing and nuclear rupture when exposed to mechanical confinement. High-resolution imaging indicates that WASp drives the formation of protective perinuclear actin structures in response to confinement. Functionally, WASp null macrophages respond to mechanical confinement by inducing a transcriptional profile consistent with the release of immunogenic DNA in the cytosol. The proinflammatory state of mechanically confined WASp-deficient macrophages depends, in part, on the cGAS-STING pathway of cytosolic DNA sensing. Together, these data uncover a WASp-dependent mechanism to restrict activation of inflammatory signalling in tissue macrophages and provide hints to target unabated inflammation in Wiskott-Aldrich Syndrome.
Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis.
Project description:The pleiotropic RTK Kit can provide cytoskeletal signals that define cell shape, positioning and migration, but the underlying mechanisms are less well understood. Here we provide evidence that Kit signals through WASP (Wiskott-Aldrich Syndrome Protein), the central hematopoietic actin nucleation- promoting factor and regulator of the cytoskeleton. KL-mediated gene expression in WT and WASP-deficient BMMCs was compared and revealed that approximately 30% of all Kit-induced changes were WASP-dependent. The results indicate that Kit signaling through WASP is necessary for normal Kit-mediated filopodia formation, cell survival and gene expression and provide new insight in the mechanism how WASP exerts a strong selective pressure in hematopoiesis.