Project description:The project consisted of single and double knockdown experiments involving forty-five genes in the human squamous carcinoma cell culture line A431. During the knockdown phase, the cells were transfected with siRNAs which decreased target gene expression. After 72 hours the cells were deemed to have reached a new steady-state and approximate cell counts were calculated using resazurin. At this point the cells were lysed using CelluLyser, and a proprietary RNA spike was added to each sample relative to the cell count to act as a reference gene for the qPCR analysis. Then cDNA was prepared from the RNA and preamplified in preparation for the high-throughput qPCR screening. The transcript profiles, with respect to the forty-five target genes, were determined using TaqMan assays on Fluidigm's Biomark platform using 96x96 Dynamic Array IFCs.
Project description:Transcriptional response of the QS-negative D. shibae strain ∆luxI1 towards externally added 500nM autoinducer (AI) 3-oxo-C14-HSL over a time period of 3 hours.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively-bound BATF and IRF4 contribute to initial chromatin accessibility, and with STAT3 initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple datasets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease. 143 RNA-seq, 83 ChIP-seq, 65 ChIP-seq controls, and 16 FAIRE-seq
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.
Project description:Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.