DNA-binding specificity of the CYS3 transcription factor of Neurospora crassa defined by binding-site selection

The CYS3 transcription factor is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of a coordinately regulated group of genes involved in the acquisition and utilization of sulfur in Neurospora crassa. An approach of using binding-site selection from random-sequence oligonucleotides was used to define CYS3-binding specificity. The derived consensus-binding site of ATGGCGCCAT defines a symmetrical sequence (half-site A T G/t G/a C/t) that resembles that of other bZIP proteins such as CREB and C/EBP. By comparison, CYS3 shows a greater range of binding to a central core of varied Pur–Pyr–Pur–Pyr sequences than CREB as determined by gel shift assays. The derived CYS3 consensus binding sequence was further validated by demonstrating in vivo sulfur regulation using a heterologous promoter construct. The CYS3-binding site data will be useful for the genome-wide study of sulfur-regulated genes in N. crassa, which has served as a model fungal sulfur control system.