CO2-cAMP-Responsive cis-Elements Targeted by a Transcription Factor with CREB/ATF-Like Basic Zipper Domain in the Marine Diatom Phaeodactylum tricornutum

Abstract

Abstract Expression controls of the carbon acquisition system in marine diatoms in response to environmental factors are an essential issue to understand the changes in marine primary productivity. A pyrenoidal β-carbonic anhydrase, PtCA1, is one of the most important candidates to investigate the control mechanisms of the CO2 acquisition system in the marine diatom Phaeodactylum tricornutum. A detailed functional assay was carried out on the putative core regulatory region of the ptca1 promoter using a β-glucuronidase reporter in P. tricornutum cells under changing CO2 conditions. A set of loss-of-function assays led to the identification of three CO2-responsive elements, TGACGT, ACGTCA, and TGACGC, at a region −86 to −42 relative to the transcription start site. Treatment with a cyclic (c)AMP analog, dibutyryl cAMP, revealed these three elements to be under the control of cAMP; thus, we designated them, from 5′ to 3′, as CO2-cAMP-Responsive Element1 (CCRE1), CCRE2, and CCRE3. Because the sequence TGACGT is known to be a typical target of human Activating Transcription Factor6 (ATF6), we searched for genes containing a basic zipper (bZIP) region homologous to that of ATF6 in the genome of P. tricornutum. Gel-shift assays using CCRE pentamers as labeled probes showed that at least one candidate of bZIP proteins, PtbZIP11, bound specifically to CCREs. A series of gain-of-function assays with CCREs fused to a minimal promoter strongly suggested that the alternative combination of CCRE1/2 or CCRE2/3 at proper distances from the minimal promoter is required as a potential target of PtbZIP11 for an effective CO2 response of the ptca1 gene.