Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation

Abstract

The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1) and Lcr2 (Low-CO 2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in Chlamydomonas.

A transcriptional regulatory circuit for the photosynthetic acclimation of microalgae to carbon dioxide limitation

“…In agreement with previous work (Berger et al, 2014), which clearly demonstrated an activation of the NAB1 promoter by carbon dioxide depletion via reporter assays, a transcriptome study revealed changes in NAB1 transcript abundance dependent on carbon dioxide supply (Winck et al, 2013).…”

“…Six of these motifs are present in the NAB1 promoter (Supplementary Figure S2, Supplementary Table S2), but none of them is located within the 152 bp region upstream of the translation start site. Moreover, according to Winck and co-workers, NAB1 (UniProt KB ID 126810) was contained in a cluster of early responding genes and mRNA levels increased about 7-fold one hour after the onset of carbon dioxide deprivation (Winck et al, 2013). NAB1 accumulation in response to carbon dioxide limitation requires LCRF ( Figure 3C) and results in a decline of LHCBM6 protein levels after six hours ( Figure 4F).…”

“…An in silico analysis regarding the presence of known cis-regulatory elements on the NAB1 promoter was performed (Supplementary Figure S2), using the databases PLACE (Higo et al, 1999) and PlantCARE (Lescot et al, 2002). In addition, a recent transcriptome study (Winck et al, 2013) was considered, in which transcription factors and regulators responding to changes in carbon dioxide levels were identified. Besides, the authors could also identify ten sequence motifs and respective motif combinations in promoters regulated by low carbon dioxide.…”

“…The databases PLACE (http://www.dna.affrc.go.jp/PLACE; Higo et al, 1999) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html; Lescot et al, 2002) were used to search for known cis-regulatory element within the 1548 bp fragment upstream of the NAB1 translation start site. Furthermore, motifs which are connected to CO 2responsiveness as identified in a transcriptome study (Winck et al, 2013), were taken into account. Additionally, PlantRegMap (http://plantregmap.cbi.pku.edu.cn/; (Jin et al, 2017)) was used to screen the NAB1 promoter for putative transcription factor binding sites.…”

“…Circuit for algal light capture control 4 Although nuclear gene expression modulation in response to carbon dioxide depletion is evident (Fang et al, 2012;Winck et al, 2013), information on the molecular mechanisms underlying these transcriptomic changes is limited (Wang et al, 2015). In general, only a small number of nuclear Chlamydomonas promoters have already been studied in the context of photosynthetic acclimation responses (Sawyer et al, 2015;Maruyama et al, 2014;Winck et al, 2013;Shao et al, 2008).…”