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

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.

Transcript level coordination of carbon pathways during silicon starvation‐induced lipid accumulation in the diatom Thalassiosira pseudonana

“…(2015) was not recovered as a single cluster in our analysis, several of these genes were coexpressed in silicon‐starved T. pseudonana, including an isoform of δ‐carbonic anhydrase (Thaps3_233), and two bestrophins (Thaps3_4819, Thaps3_4820), which are predicted to be chloroplast‐localized, up‐regulated at the protein and transcript levels under low CO 2 conditions, and hypothesized to play a role chloroplast inorganic carbon supply (Kustka et al ., 2014; Hennon et al ., 2015). The promoters of these genes are reported to contain potential cis‐ regulatory motifs which confer sensitivity to CO 2 ‐induced shifts in intracellular cAMP concentrations (CCREs; Ohno et al ., 2012; Hennon et al ., 2015). Although these genes did not cluster with ACCase chl , they were expressed with a very similar pattern (Fig.…”

Light and CO2/cAMP Signal Cross Talk on the Promoter Elements of Chloroplastic β-Carbonic Anhydrase Genes in the Marine Diatom Phaeodactylum tricornutum

“…The function of the CO 2 -responsive ptca1 promoter (Pptca1) has been studied in detail (Harada et al, 2006;Ohno et al, 2012). Three cis-elements denoted CO 2 / cAMP-responsive elements (CCRE1, CCRE2, and CCRE3), which are tandemly aligned between 286 and 242 relative to the transcription-start site, are critical motifs for Pptca1 to respond to changes in CO 2 concentration via the second messenger, cAMP (Ohno et al, 2012).…”

“…Three cis-elements denoted CO 2 / cAMP-responsive elements (CCRE1, CCRE2, and CCRE3), which are tandemly aligned between 286 and 242 relative to the transcription-start site, are critical motifs for Pptca1 to respond to changes in CO 2 concentration via the second messenger, cAMP (Ohno et al, 2012). CCRE1, CCRE2, and CCRE3 are an analogous sequence (TGACGT/C) and located in an inverted direction to each other with 12 to 15 bps of spacer sequences (Ohno et al, 2012). CCRE2 is the core element, which works with either CCRE1 or CCRE3, suggesting that two elements possessing the complementary sequence may cooperate to recruit specific transcription factors (Ohno et al, 2012).…”

“…CCRE1, CCRE2, and CCRE3 are an analogous sequence (TGACGT/C) and located in an inverted direction to each other with 12 to 15 bps of spacer sequences (Ohno et al, 2012). CCRE2 is the core element, which works with either CCRE1 or CCRE3, suggesting that two elements possessing the complementary sequence may cooperate to recruit specific transcription factors (Ohno et al, 2012). Indeed, a basic zipper (bZIP) protein PtbZIP11, which resembles the mammalian cAMP-responsive element-binding protein/activation transcription factor, is detected as a Trans-acting factor, which specifically binds to CCREs (Ohno et al, 2012).…”

“…CCRE2 is the core element, which works with either CCRE1 or CCRE3, suggesting that two elements possessing the complementary sequence may cooperate to recruit specific transcription factors (Ohno et al, 2012). Indeed, a basic zipper (bZIP) protein PtbZIP11, which resembles the mammalian cAMP-responsive element-binding protein/activation transcription factor, is detected as a Trans-acting factor, which specifically binds to CCREs (Ohno et al, 2012). However, the upstream signal transduction mechanism of the diatom CO 2 -response is still unclear, and factors controlling the Pptca1 promoter activity await further investigations.…”