Locating the DNA Site
The E. coli melAB genes encode proteins that are necessary for transport and metabolism of the disaccharide, melibiose. Expression of this protein is dependent on the transcription activator, MelR, which is encoded by the adjacent melR gene.
Previous studies have shown that transcription from the melAB promoter is activated by MelR and have focused on using biochemistry to understand the mechanism of activation.
OGT's probe design pipeline was used to fabricate the E. coli genomic DNA 22,000 feature microarray. The technology optimizes selection for base composition and minimal homology within the E. coli genome. Probes were selected with an average spacing of one probe per 230 base pairs throughout the MG1655 genome and were all 60 bases in length.
The position of the melR binding site (located from bases 4339356 to 4339373 of the E. coli genome) has been previously characterized using Affymetrix ChIP microarrays1. This method required the use of PCR amplification, which we believe could potentially cause a bias, particularly with more complex mixtures of ChIP DNA where other transcription factors, e.g., global regulators, may be present that bind to more than one site in the genome.
In this study, ChIP was carried out without PCR amplification1. Briefly, in vivo cross-linking of bacterial nucleoprotein was initiated by adding 1% formaldehyde to cultures and, after 20 minutes, cross-linking was quenched by adding 0.5 M glycine. Cells were harvested by centrifugation before washing and resuspending in lysis buffer at 37C for 30 minutes.
Following lysis, immunoprecipitation buffer was added and cellular DNA was sheared by sonication to an average size of 5001,000 bp. Samples were incubated with protein A/G beads and immunoprecipitated using antibodies to MelR; immunoprecipitation experiments without antibodies were also conducted as negative controls. Immunoprecipitated complexes were removed from the beads and samples were decross-linked.
The ChIP DNA was labeled by Klenow random priming, incorporating Cy3-dCTP or Cy5-dCTP (GE Healthcare). ChIP DNA extracted from wild type strain MG1655 was labeled with Cy3, while ChIP DNA isolated from a melR deleted strain was labeled with Cy5.
The labeled DNA was then spun through an Autoseq column (GE Healthcare). Hybridization was carried out in an Agilent microarray chamber using OGT buffer at 55C over 60 hours. For data collection, the arrays were scanned on an Agilent microarray scanner and signal quantitation of the spots was carried out using version 7.1 of the Image Analysis and Feature extraction software from Agilent; the background was subtracted using local background.
The Cy5/Cy3 intensity ratio was calculated for each spot and plotted against the corresponding position on the E. coli MG1655 chromosome. Data were visualized using an in-house developed ChIP browser, allowing the microarray data generated to be viewed according to its relative gene position (Figure 1).
Several probes on the arrays corresponding to an E. coli genomic position between 4338913 and 4339725 had a Cy3/Cy5 ratio significantly above background (Figure 2), indicating that MelR binds to this region of the genomic DNA, which is the position of the known E. coli MelR binding site.