Journal: Nature Communications

Article Title: A TetR-family transcription factor regulates fatty acid metabolism in the archaeal model organism Sulfolobus acidocaldarius

doi: 10.1038/s41467-019-09479-1

Figure Lengend Snippet: FadR Sa interacts with four genomic loci in the Saci_1103 - Saci_1126 gene cluster. a Overview of the genomic binding profile of FadR Sa as monitored by ChIP-seq (IP = immunoprecipitated sample). A zoomed image of this profile is shown for the genomic region encompassing the Saci_1103-Saci_1126 gene cluster (corresponding to genomic coordinates 903,000–932,000), with indication of the four clearly visible peaks (numbered 1–4). Below the profile, a schematic representation of the genomic organization of the Saci_1103 - Saci_1126 gene cluster is shown with indication of the ChIP-seq peak summit locations and of the transcription start sites as detected in the transcriptomic analysis in ref. . b ChIP-qPCR experiment with targeted quantification of enrichment for peaks 1 and 2 (given their close proximity to each other, these are assayed within a single fragment representing the Saci_1106 / Saci_1107 intergenic region), peak 3 and peak 4. Fold enrichment is expressed relative to a genomic region within the ORF of Saci_1336 that was shown not be bound by FadR Sa in the ChIP-seq profile. Error bars represent standard deviations of biological duplicates. c Sequence logo of the FadR Sa binding motif representing MEME predictions of ChIP-seq enriched sequences. d Electrophoretic mobility shift assays of FadR Sa binding to radiolabeled DNA probes of about 500 bp representing the ChIP-seq peaks identified in the Saci_1103 - Saci_1126 gene cluster (see panel ( a )). Molar protein concentrations are shown above each autoradiograph, whereas populations of free DNA (F) and FadR Sa -bound DNA (B1 and B2) are indicated with an arrowhead. Apparent K D and Hill coefficient ( n ) calculations are based on densitometric analysis of free DNA bands followed by binding curve analysis (Supplementary Figure )

Article Snippet: Primers were designed with Primer3 Plus software (Supplementary Data ) and were chosen to amplify fragments around the ChIP-seq peak summit regions and with a length between 150 and 200 bp. qPCR was performed with a My-iQ TM Single color Real-time PCR system (Bio-Rad) and GoTaq qPCR Master Mix (Promega) was done with thermal cycling conditions: 10 min at 94 °C and 40 cycles of 30 s at 94 °C and 30 s at 60 °C.

Techniques: Binding Assay, ChIP-sequencing, Immunoprecipitation, ChIP-qPCR, Sequencing, Electrophoretic Mobility Shift Assay, Autoradiography