Calcineurin responsive zinc-finger-1 binds to a unique promoter sequence to upregulate neuronal calcium sensor-1, whose interaction with MID-1 increases tolerance to calcium stress in Neurospora crassa

Abstract

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca²⁺ stress in Neurospora crassa. Increasing concentration of Ca²⁺ increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca²⁺ concentrations. These results suggested the regulation of NCS-1 function through calcineurin- CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that the CRZ-1 binds specifically to an 8 bp sequence 5'-CCTTCACA-3' in the ncs-1 promoter 216 bp upstream of the ATG start codon. We also showed that NCS-1 binds to the Ca²⁺ permeable channel MID-1 for tolerance to Ca²⁺ stress. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter, causing upregulation of NCS-1 that binds to MID-1 for tolerance to Ca²⁺ stress.