Project description:Gene expression profiles of Cbfb-deficient and control Treg cells were compared. Abstract: Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here we showed that Treg cell-specific deficiency of Cbfβ, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyper-production of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfβ heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3. Experiment Overall Design: CD4+CD25hi cells, most of which were Foxp3+ Treg cells, were isolated from Cbfb-flox/flox: Foxp3-ires-Cre (n = 3) and control Cbfb-flox/wt: Foxp3-ires-Cre (n = 3) mice. Total RNA was extracted from those purified Cbfb-deficient or control Treg cells.
Project description:Effect of FIS and H-NS on gene expression at relexed and hypernagative supercoiling level using LZ41 and LZ54 strains. LZ41 and LZ54 strains contain drug-resistant alleles of different topoisomerase genes. LZ41 strain treatment norfloxacin strongly relaxes DNA, whereas in LZ54 strain the same treatment generates high negative supercoiling (Khodursky et al., 1995, PNAS 92:11801-5; Ziechedrich et al, 1997, Genes Dev. 11:2580-92).
Project description:Effect of supercoiling level on gene expression using LZ41 and LZ54 strains containing drug-resistant alleles of different topoisomerase genes. LZ41 strain treatment with norfloxacin strongly relaxes DNA, whereas in LZ54 strain the same treatment generates high negative supercoiling (Khodursky et al., 1995, PNAS 92:11801-5; Ziechedrich et al, 1997, Genes Dev. 11:2580-92).
Project description:Growth curves wt and hns strains in rich dYT medium. Sample in Mid-exponential phase (ME), Transition to Stationary (TS) and Late Stationary phase (LS).
Project description:Conditioned medium (CM) and extracellular vesicles (EV) from human Adipose-derived Stem/stromal cells (ASC) and Dermal fibroblasts (DF) can represent valid cell substitutes in regenerative medicine and other therapeutic applications. Whether one of the two cell products should be preferred over the other is still under debate and no direct comparison of their proteome has been reported yet. Here, we apply a comprehensive quantitative proteomics approach to explore the protein composition of conditioned medium and EV obtained from the two cell types. We identified 1977 proteins by LC-MS/MS proteomic analysis. Unsupervised clustering analysis and PCA clearly distinguished CM and EV as separate groups, regardless the cell source. We identified 68 and 201 proteins that were more abundant or exclusively detectable in CM and EV, respectively. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation-related factors. Moreover, the analysis of ASC and DF secretomes (both CM and EV) revealed the presence of cell type-specific factors. ASC-CM and -EV carried factors involved in ECM organization (hyaluronan and glycosaminoglycan metabolism) and immunological regulation (e.g. macrophage and IkB/NFkB signaling regulation), respectively. On the other hand, DF-CM and –EV were both enriched in epithelium development associated factors, whilst DF-CM in proteins involved in cellular processes regulation and -EV in Wnt signaling factors. In conclusion, this comprehensive proteomic analysis of the secretome components of ASC and DF, provides evidence of a different protein composition between CM and EV and of the presence of cell type-specific bioactive mediators suggesting their specific future use as advanced therapy medicinal products.
Project description:In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here, we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is a target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. Corresponding variations in protein levels led us to hypothesize that coordinated RNA decay represents another level in the hierarchical methionine biosynthesis control network to adjust methionine biosynthesis enzyme amounts to current metabolic requirements.
Project description:The ability to grow at host temperature is a critical trait for most pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are a class of soil fungi that undergo a dramatic change in cell shape and virulence gene expression in response to host temperature. Here we elucidate a complex temperature-responsive network in H. capsulatum, which switches from an environmental filamentous form to a pathogenic yeast form. We dissect the circuit driven by three regulators that control yeast-phase growth, and demonstrate that these factors, including two deeply conserved Velvet family proteins of unknown function, associate with DNA. We identify and characterize a fourth regulator of this pathway, and define cis-acting motifs that recruit these transcription factors to a tightly interwoven network of temperature-responsive target genes. Our results provide the first comprehensive analysis of the complex transcriptional network that links temperature to morphology and virulence in thermally dimorphic fungi. This submission gives the expression profiling results. cDNA from each ryp mutants and wild-type controls was labeled with Cy5 and competitively hybridized against the Cy3-labeled pooled reference sample using H. capsulatum whole-genome 70-mer oligonucleotide microarray. For the experiments with ryp T-DNA mutants, there were 4 to 6 replicates for each strain and condition, and for the experiments with ryp knockdown strains, there were 3 to 12 replicates for each strain and condition. The T-DNA and knockdown experiments are being submitted as separate series, with samples further divided based on G217B platform version.
Project description:The ability to grow at host temperature is a critical trait for most pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are a class of soil fungi that undergo a dramatic change in cell shape and virulence gene expression in response to host temperature. Here we elucidate a complex temperature-responsive network in H. capsulatum, which switches from an environmental filamentous form to a pathogenic yeast form. We dissect the circuit driven by three regulators that control yeast-phase growth, and demonstrate that these factors, including two deeply conserved Velvet family proteins of unknown function, associate with DNA. We identify and characterize a fourth regulator of this pathway, and define cis-acting motifs that recruit these transcription factors to a tightly interwoven network of temperature-responsive target genes. Our results provide the first comprehensive analysis of the complex transcriptional network that links temperature to morphology and virulence in thermally dimorphic fungi. This submission gives the expression profiling results. cDNA from each ryp mutants and wild-type controls was labeled with Cy5 and competitively hybridized against the Cy3-labeled pooled reference sample using H. capsulatum whole-genome 70-mer oligonucleotide microarray. For the experiments with ryp T-DNA mutants, there were 4 to 6 replicates for each strain and condition, and for the experiments with ryp knockdown strains, there were 3 to 12 replicates for each strain and condition. The T-DNA and knockdown experiments are being submitted as separate series, with samples further divided based on G217B platform version.
Project description:The ability to grow at host temperature is a critical trait for most pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are a class of soil fungi that undergo a dramatic change in cell shape and virulence gene expression in response to host temperature. Here we elucidate a complex temperature-responsive network in H. capsulatum, which switches from an environmental filamentous form to a pathogenic yeast form. We dissect the circuit driven by three regulators that control yeast-phase growth, and demonstrate that these factors, including two deeply conserved Velvet family proteins of unknown function, associate with DNA. We identify and characterize a fourth regulator of this pathway, and define cis-acting motifs that recruit these transcription factors to a tightly interwoven network of temperature-responsive target genes. Our results provide the first comprehensive analysis of the complex transcriptional network that links temperature to morphology and virulence in thermally dimorphic fungi. This submission gives the expression profiling results. cDNA from each ryp mutants and wild-type controls was labeled with Cy5 and competitively hybridized against the Cy3-labeled pooled reference sample using H. capsulatum whole-genome 70-mer oligonucleotide microarray. For the experiments with ryp T-DNA mutants, there were 4 to 6 replicates for each strain and condition, and for the experiments with ryp knockdown strains, there were 3 to 12 replicates for each strain and condition. The T-DNA and knockdown experiments are being submitted as separate series, with samples further divided based on G217B platform version.