Comment[ArrayExpressAccession] E-GEOD-47341 MAGE-TAB Version 1.1 Public Release Date 2013-06-14 Investigation Title A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen -- ChIP-chip Comment[Submitted Name] A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen -- ChIP-chip Experiment 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 chromatin immunoprecipitation results. For each of the four Ryp proteins, ChIP vs. input hybridizations were performed for three replicate immunoprecipitations from the wild-type strain and two negative control replicates from the corresponding mutant strain. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Voorhies Beyhan Voorhies Sil Person First Name Mark Sinem Mark Anita Person Email mark.voorhies@ucsf.edu Person Affiliation University of California Person Address Microbiology and Immunology, University of California, 513 Parnassus, S472, San Francisco, CA, USA Person Roles submitter Protocol Name P-GSE47341-1 P-GSE47341-3 P-GSE47341-4 P-GSE47341-2 P-GSE47341-5 Protocol Description Background subtracted probe intensities were transformed to log2 (red/green) ratios (M) and log2 (sqrt(red*green)) geometric mean intensities (A), excluding probes with intensities below background. The M,A values were fit using the LOWESS algorithm (The American Statistician 35:54) as implemented in R, and the fit curve was subtracted from the transformed data to yield lowess normalized log2 ratio values. ID_REF = VALUE = LOWESS normalized log2 ChIP enrichment ratio rBGSubSignal = Background subtracted Cy5 signal from Agilent Feature Extractor gBGSubSignal = Background subtracted Cy3 signal from Agilent Feature Extractor Both input and output DNA were amplified and labeled with fluorescent dyes (Cy3 and Cy5) using strand displacement amplification following published protocol (Methods in Enzymology 470:737). Labeled DNA samples were hybridized following Agilent's ChIP-on-chip protocol. 300 mg of ground samples were vortexed vigorously in lysis buffer [50 mM Hepes/KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate supplemented with Halt protease and phosphatase inhibitors (Pierce)] for 3 hrs with 0.5 mm glass beads to lyse the cells. Following cell lysis, lysates were sonicated in a Diagenode Bioruptor for 30 min (30 sec on, 1min off) to shear DNA. Input DNA was collected and rest of the samples was subjected to immunoprecipitation. For filamentous-phase samples (ryp mutants), lysates from three identical tubes were combined prior to antibody incubation. Each sample was incubated overnight at 4C with 20 ug of polyclonal antibodies against portion of Ryp1 (ID:2877, ELDKPFPPGEKKRA), Ryp2 (ID:237, QTNRDYPFYNGPDAKRPR), Ryp3 (ID:338, GIKIPIRKDGVKGPRGGQ), or Ryp4 (ID:8, PPPQQSLQGWSPEEW). Next day, 40 ul of Protein-A Sepharose 4B Fast Flow (Sigma-Aldrich) beads were added lysate-antibody mixture and further incubated for 2 hrs. Then, beads were collected by centrifugation, and washed 9 times. Protein-DNA complexes that are bound to the beads were collected by incubating beads at 65C with elution buffer (50 mM Tris.HCl pH8.0, 10mM EDTA, 1% SDS). Protein-DNA crosslinks in input and ChIPed samples were reversed overnight at 65C. Proteins were digested with proteinase K and DNA was purified with phenol: chloroform extraction as described (Methods in Enzymology 470:737). Slides were scanned with an Agilent scanner and raw ratios were obtained with the Agilent ChIP_107_Sep09 protocol. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name STRAIN OR LINE Experimental Factor Type strain or line Comment[SecondaryAccession] GSE47341 Comment[GEOReleaseDate] 2013-06-14 Comment[ArrayExpressSubmissionDate] 2013-05-23 Comment[GEOLastUpdateDate] 2013-06-14 Comment[AEExperimentType] ChIP-chip by tiling array SDRF File E-GEOD-47341.sdrf.txt