DISTINCT CIS-ACTING ELEMENTS MEDIATE CLOCK, LIGHT, AND DEVELOPMENTAL REGULATION OF THE NEUROSPORA-CRASSA EAS (CCG-2) GENE

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydroph obin, is transcriptionally regulated by the circadian clock. In additi on, eas (ccg-2) is positively regulated by light and transcripts accum ulate during asexual development. To sort out the basis of this comple x regulation, deletion analyses of the eas (ccg-2) promoter were carri ed out to localize the cis-acting elements mediating clock, light, and developmental control, The primary sequence determinants of a positiv e activating dock element (ACE) were found to reside in a 45-bp region , just upstream from the TATA box. Using a novel unregulated promoter- reporter system developed for this study, we show that a 68-bp sequenc e encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N, crassa protein extracts that recognize a nd bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmen tal control are identified and shown not to be required for clock-regu lated expression of eas (ccg-2). The distinct nature of the ACE valida tes its use as a tool for the identification of upstream regulatory fa ctors involved in clock control of gene expression.