A novel activation domain is essential for CIA5-mediated gene regulation in response to CO2 changes in Chlamydomonas reinhardtii

The inducible CO2 concentrating mechanism (CCM) of microalgae is essential for their acclimation to highly variable aquatic inorganic carbon levels. The key transcriptional regulator, CIA5, affects expression of thousands of genes in Chlamydomonas reinhardtii, but the molecular characteristics of this important protein are poorly understood. This study identifies a functional activation domain in CIA5 and demonstrates the functionality of a mini-CIA5 including only the zinc-binding domain and the identified activation domain. A highly conserved 130aa region from CIA5 exhibits auto-activation in yeast and appears responsible for the markedly slow migration of CIA5 when analyzed by SDS-PAGE. This 130aa region or either half of this region also effectively replaced the activation domain of a modified designer Transcription Activator-like Element (dTALE) in targeted activation of an endogenous Chlamydomonas gene. Additionally, a mini-CIA5 combining the conserved zinc-binding domain with the 130aa putative activation domain complemented the growth phenotype of the cia5 mutant and triggered CO2-regulated gene expression patterns similar to wild-type cells or cia5 complemented with the full-length CIA5. Although the mini-CIA5 complementation did not fully restore wild-type growth rates or full gene induction/repression amplitudes, especially in very low CO2, this newly identified activation domain combined with the previously described zinc-binding domain are demonstrated to be the key essential components of CIA5 that permit rapid CIA5-mediated responses to changes in CO2 concentrations. •This paper demonstrates that a region of the CIA5 protein from approximately amino acid 428 through amino acid 557 acts as an activation domain. CIA5 is a “master regulator” of the CO2-concentrating-mechanism in Chlamydomonas reinhardtii and thus plays an important role in this model microalga’s adaptation to limiting CO2 growth conditions. In addition to demonstrating that this activation domain is functional in Chlamydomonas, the paper also demonstrates it functions as an activation domain in yeast. The paper also demonstrates that a “mini-CIA5” protein comprising only this activation domain plus a DNA-binding domain are sufficient to largely complement a cia5 mutant, indicating these two domains are the only essential domains needed for CIA5 function as a transcription regulator.