Abstract
BackgroundThe interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner.ResultsWe facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor.ConclusionOur method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.
Highlights
The interaction between a transcription factor and DNA motif is an important regulatory step in gene regulation
Transcription factors (TFs) are regulatory proteins that interact with specific DNA sequences to control gene expression
Design of the Q9-protein binding microarray (PBM) We designed a PBM, which we refer to as Quadrupled 9-mer Protein Binding Microarray (Q9-PBM), in such a way that target probes are synthesized as quadruples of all possible 9-mer combinations
Summary
The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Transcription factors (TFs) are regulatory proteins that interact with specific DNA sequences to control gene expression. The DNA binding domains of TFs bind to specific upstream sequences (cis-acting elements) of target genes and modulate the transcription process. ProteinDNA binding properties have been investigated by traditional procedures, such as the Electrophoretic Mobility Shift Assay (EMSA) and filter binding assay [1,2] These methods are labor-intensive and are restricted (page number not for citation purposes). Comprehensive genome-wide methods, along with the availability of whole-genome sequences and advances in microarray technology, have been developed to characterize protein-DNA binding specificities [3]. ChIP-chip might be applicable under the restrictions of the antibodies available for each protein
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