In our contribution we will characterise multitude of different interactions between DNA binding proteins (transcription factors) and regulatory elements encoded in diverse DNA stretches to gain an improved understanding of the transcriptional regulation. In a highly parallel fashion these interaction will be characterised by using our in house well-established protein microarrays and the recently established double-stranded DNA microarray technique.
Fabrication of human transcription factor protein microarrays. Protein microarrays presenting transcription factors will be fabricated with our in house optimised high-end microarrayers. Purified proteins will be transferred into 384 well microtitre plates and transferred by high precission pins onto microarray blanks. All proteins will be spotted in at least five replicates together with suitable guide dots and dilution series of markers for robust statistical evaluations. The slides will be spotted on demand and stored at 4 oC in a humid environment (>60 %) for short periods of time (< 1 month).
Fabrication of dsDNA microarrays Microarrays presenting dsDNA in an oriented fashion accessible for proteins will be fabricated according to protocols already established. We will amplify promoters from genomic DNA or BAC/ YAC clones by PCR and increase the amount of the target sequence in a second re-amplification step relying on universal primers. One universal primer will be 5’ biotinylated for a directed immobilisation of the PCR products on streptavidin coated microarray substrates. Following the purification of PCR products the promotor products will be spotted as double-stranded PCR products in high density on streptavidin coated substrates. The slides will be directly used for the interaction screenings.
Screen for protein-DNA interaction using protein and dsDNA microarrays Immobilized proteins will be tested for DNA binding. Protein microarrays will be incubated with purified PCR fragments in the presence of competitor DNA to compete unspecific DNA binding to the proteins. All DNA sequences will be incubated on the transcription factor microarrays in the same concentration. Retained DNA will be detected with a microarray scanner. The sample (PCR fragment) will be spiked with a specific DNA that will only bind to the positive. Thereby we will ensure comparability between different slides. Affinity purified transcription factors will be tested for specific DNA binding. The proteins will be diluted to the same working concentration and incubated with the immobilised double stranded DNA. After incubation and stringent washing steps proteins that have bound specifically to their target sequence will be detected with labelled antibodies against the affinity tag or by direct labelling of the transcription factors. The identified transcription factor–target DNA interactions will be cross checked with knowledge based databases in close co-operation with project 5.1.
