Project description:To induce chronic IFN-I activation, we injected wildtype and Ifnar1–/– mice with five treatments of DMXAA, a STING agonist, followed by snRNA-seq analysis of hippocampal tissues. In wildtype mice, we showed that microglia had the strongest IFN response to STING activation of all cell types. Ablation of interferon alpha and beta receptor 1 (IFNAR1) abolished microglial IFN-I response and suppression of neuronal MEF2C transcriptional network
Project description:In bacteria, the biosynthesis of cysteine is accomplished by two enzymes that are encoged by the cysK and cysM genes. CysM is also able to incorporate thiosulfate to produce S-sulfocysteine. In plant cells, the biosynthesis of cysteine occurs in the cytosol, mitochondria and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of cs26 suppressed the S-sulfocysteine synthase activity that was detected in wild type; furthermore, the mutants exhibited a growth phenotype, but penetrance depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants), as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify ROS, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our finding reveals that S-sulfocysteine and the activity of S-sulfocysteine synthase play an important role in chloroplast function and are essential for light-dependent redox regulation within the chloroplast.
Project description:In bacteria, the biosynthesis of cysteine is accomplished by two enzymes that are encoged by the cysK and cysM genes. CysM is also able to incorporate thiosulfate to produce S-sulfocysteine. In plant cells, the biosynthesis of cysteine occurs in the cytosol, mitochondria and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of cs26 suppressed the S-sulfocysteine synthase activity that was detected in wild type; furthermore, the mutants exhibited a growth phenotype, but penetrance depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants), as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify ROS, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our finding reveals that S-sulfocysteine and the activity of S-sulfocysteine synthase play an important role in chloroplast function and are essential for light-dependent redox regulation within the chloroplast. Using the Affymetrix ATH1 GeneChips, we performed a comparative transcriptomic analysis on leaves of the cs26 and wild type plants under two different photoperiod conditions. Wild type and cs26 mutant plants were grown on soil under a long-day photoperiod (LD) or under a short-day photoperiod (SD). Total RNA was extracted from the leaves of 3-week-old plants grown under identical LD conditions, and from the leaves of 5-week-old plants grown under identical SD conditions. Three biological replicates were performed for each sample and hybridized to the chips. We made two different comparisons to classify the differently expressed genes in the mutant plant: cs26 leaves under LD versus wild-type leaves under LD and cs26 leaves under SD versus wild-type leaves under SD.
Project description:We have isolated, analyzed and compared the RNA content of various cell compartments including total RNA, nucleolplasmic RNA and nucleolar RNA. In addition, cells have been subjected to various treatment to specifically inhibit each of the RNA polymerases I, II and III, as well as protein synthesis. Cells were also treated with antisense oligos to induce the degradation of specific RNA transcripts. The whole RNA content of the cells after these specific treatments was isolated and sequenced.
Project description:Biallelic genetic variants in N-acetylneuraminic acid synthase (NANS), a critical enzyme in endogenous sialic acid biosynthesis, are clinically associated with neurodevelopmental disorders. However, the mechanism underlying the neuropathological consequences has remained elusive. Here, we discovered that NANS mutation resulted in absence of both sialic acid and protein polysialylation in the cortical organoids and significantly reduced the proliferation and expansion of neural progenitors. NANS mutation dysregulated neural migration and differentiation, disturbed synapse formation, and weakened neuronal activity. Single-cell RNA sequencing revealed that NANS loss-of-function markedly altered transcriptional programs involved in neuronal differentiation and ribosomal biogenesis in various neuronal cell types. Similarly, Nans heterozygous mice exhibited impaired cortical neurogenesis and neurobehavioral deficits. Collectively, our findings reveal a crucial role of NANS-mediated endogenous sialic acid biosynthesis in regulating multiple features of human cortical development, thus linking NANS mutation with its clinically relevant neurodevelopmental disorders.
Project description:Cells regulate gene expression using a complex network of signaling pathways, transcription factors and promoters. To gain insight into the structure and function of these networks we analyzed gene expression in single and multiple mutant strains to build a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast. Our model reveals that the Hog1 and general stress (Msn2/4) pathways interact, at both the signaling and promoter level, to integrate information and create a context-dependent response. Keywords: Stress response network analysis using genetically modified cells