Regulatory Targeting Genes of RISBZ1, a Seed Specific Transcription Factor, with Protein Binding Microarray

The survey of binding affinity motifs of a particular transcription factor is important to understand the developmental specific gene expression and regulatory networks of an organism. Protein-binding microarrays (PBMs) provide useful predictions for exploring the transcriptional regulatory code in a genome-wide manner. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, named Q9-UPBM, consists of 274,480 probes. We analyzed the binding motifs of RISBZ1 protein, an endosperm specific basic leucine zipper transcription factor, to determine targeting genes of a transcription factor on the Q9-UPBM. RISBZ1 has been known to interact with the GCN4 motif (TGAGTCA) to regulate seed storage proteins. This experiment shows DsRed-fused RISBZ1 expressed in E.coli could bind to CCACGTCA and GGATGAC motifs as well as GCN4 motif known as binding motif. To compare the effect of posttranslational modification, the DsRed-fused RISBZ1 protein was also produced in rice callus. The binding motif of this recombinant RISBZ1 protein was determined as CCACGTCAG site, but not GCN4 motif. The Q9-UPBM experiments using the protein expressed in callus suggested more specific binding motif, a nonamer. We found 298 genes correlated with RISBZ1 in rice through RiceArrayNet, which provides co-expression information between genes in terms of correlation coefficients from accumulated 300k rice expression Microarray (http://bioinfo.mju.ac.kr/arraynet/rice300k_2011/). In combination of the binding motifs and correlated genes, the target genes of RISBZ1 transcription factor were 4 positive regulated target genes - Os02g0569400 (Cytochrome P450 family protein), Os02g0569400 (Cyclin-like F-box domain protein), Os05g0499100 (26 kDa globulin), Os08g0556000 (YT521-B-like protein family protein), and 2 nagative regulated target genes - Os02g0794400 (Initiation factor 3 family protein), Os10g0509200 (PAP fibrillin family protein). Our results will provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the gene expression during development.