Identification of a gene controlling levels of the copper response regulator 1 transcription factor in Chlamydomonas reinhardtii
Oxygen prevents hydrogen production in Chlamydomonas (Chlamydomonas reinhardtii), in part by inhibiting the transcription of hydrogenase genes. We developed a screen for mutants showing constitutive accumulation of iron hydrogenase 1 (HYDA1) transcripts in normoxia. A reporter gene required for ciliary motility placed under the control of the HYDA1 promoter conferred motility only in hypoxia. By selecting for mutants able to swim even in normoxia, we obtained strains that constitutively express the reporter gene. One identified mutant was affected in a gene encoding an F-box protein 3 (FBXO3) that participates in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1 (constitutive expression of hydrogenases and copper-responsive genes), triggers the upregulation of genes known to be targets of copper response regulator 1 (CRR1), a transcription factor involved in the nutritional copper signaling pathway and in the hypoxia response pathway. CRR1 was required for upregulating the HYDA1 reporter gene expression in response to hypoxia and for the constitutive expression of the reporter gene in cehc1-1 mutant cells. The CRR1 protein, normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 facilitates CRR1 degradation. Our results describe a previously unknown pathway for CRR1 inhibition and possibly other pathways leading to complex metabolic changes. Overall design: RNA-seq was carried out for three strains available at the Chlamydomonas Resource Center (see CC accession numbers in parentheses). The experiment compared the transcriptome of wild-type cells (strain CC-4507) with that of cells carrying a mutation in the CEHC1 gene (JGI ver6.1 Cre01.g005900) (strain CC-4509).  To confirm transcripts regulated by the CEHC1 gene, we also obtained the transcriptome of a strain in which the cehc1-1 mutant phenotype was rescued by transformation with the wild-type CEHC1 gene (strain CC-5386).