Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs. RNAseq and ChIPseq upon knockout mutants and sild type growing on various carbon sources to determin the role of the transcription factors: CLR-1, CLR-2, and XLR-1 in plant cell wall degradation
Project description:In bacteria, Crp-Fnr superfamily transcription factors mediate 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) signaling. The CRP-like protein Clr of the soil-dwelling and plant-symbiotic α-proteobacterium Sinorhizobium meliloti was previously shown to activate target promoters in both its cAMP- and cGMP-bound states (Krol et al., Microbiology 62:1840–1856, 2016). In order to further characterize the overall regulon of Clr in S. meliloti Chromatin Immuno Precipitation DNA-Sequencing (ChIP-Seq) experiments were performed with C-terminally FLAG tagged Clr under four different growth conditions, namely growth in TY complex medium and MOPS minimal medium, each supplemented with either cAMP or cGMP. For each condition, the respective immunoprecipitated (IP) and non-immunoprecipitated (control) samples were analyzed and compared to locate genomic positions in which Clr-DNA binding occurs. In combining ChIP-Seq with Electrophoretic Mobility Shift Assays and promoter-probe assays we expanded the list of known Clr-regulated target promoters and showed that virtually all of these promoters containing a palindromic Clr binding site (CBS) motif are activated both by Clr•cAMP and Clr•cGMP.
Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs.
Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs.
Project description:The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f−/−) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f−/− mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f−/− mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1−/−Clr-f−/− background reveals that Clr-f−/− mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.
Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs. RNAseq and ChIPseq was performed upon knockout mutants and wild type strains growing on various carbon sources to determin the role of the transcription factors CLR-1, CLR-2, and XLR-1 in plant cell wall degradation
Project description:Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and plays key roles in biological carbon cycle. Although cellulases induction recently described in some saprobic filamentous fungi, regulation of cellulase transcription has not been studied thoroughly. Here, we identified and characterized the novel cellulase regulation factors clr-4 in Neurospora crassa and its ortholog Mtclr-4 in Myceliophthora thermophila. Deletion of clr-4 and Mtclr-4 displayed similarly defective phenotypes in cellulolytic enzymes production and activities. Transcriptomics analysis of Δclr-4/ΔMtclr-4 revealed down-regulation of not only encoding genes of (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1), but also the key genes of cAMP signaling pathway such as adenylate cyclase cr-1. Consistently, the significant decreased levels of intracellular cAMP were observed in Δclr-4/ΔMtclr-4 compared to wild-type during cellulose utilization. Electrophoretic mobility shift assays (EMSA) verified that CLR-4 could directly bind to the promoter regions of adenylyl cyclase (Nccr-1) and cellulose regulator clr-1, while MtCLR-4 bind to upstream regions of adenylyl cyclase Mtcr-1 and biomass deconstruction regulators Mtclr-2 and Mtxyr-1. Concluded, the novel cellulase expression regulators (CLR-4/MtCLR-4) findings here significantly enrich our understanding of the regulatory network of cellulose degradation and provide new targets for industrial fungi strain engineering for plant biomass deconstruction in biorefinery.