Regulation of gene expression in the dinoflagellate Lingulodinium polyedrum

Dinoflagellates are unicellular eukaryotes found in both marine and freshwater environments. They are best known for causing toxic blooms called ‘red-tides’, for their symbiosis with corals, and for their important contribution to carbon fixation in the ocean. On a more molecular level, they are also known for their unique nuclear characteristics, as they generally have huge amount of DNA found in chromosomes that are permanently condensed and packaged into liquid crystalline forms instead of nucleosomes. Nuclear-encoded genes are often present in multiple copies and arranged in tandem, and no putative promoter elements including the conserved TATA box, have yet been observed. The unique organization of dinoflagellate chromatin suggests different strategies may be required to regulate gene expression in these organisms. In this study, I have started to address this problem using the photosynthetic dinoflagellate Lingulodinium polyedrum as a model. L. polyedrum is of particular interest because it shows a number of circadian (daily) rhythms. To date, all circadian changes in gene expression studied are regulated at a translational level. I have used transcriptomic, proteomic and phosphoproteomic approaches along with biochemical studies to provide insight into the gene regulatory mechanisms in dinoflagellates, with particular emphasis on the importance of phosphorylation in the L. polyedrum circadian system.    The absence of histone proteins and nucleosomes is a hallmark of the dinoflagellates. Using high throughput RNA-seq technology, I found complete set of sequences encoding the core histones as well as sequences encoding histone-modifying enzymes in L. polyedrum. Thus L. polyedrum expresses conserved histone transcripts, although levels of proteins are still below what can be detected using immunoblotting studies.   Using the de novo assembly algorithm the RNA-seq data was used to generate a transcriptome. This transcriptome, a list of genes expressed by L. polyedrum, has been extensively characterized. First, homology based sequence searches were used to classify the transcripts in gene ontology (GO) categories, and this analysis revealed a reduced number of transcription factor types and a surprising predominance of sequences containing a cold shock domain. Alignments of reads from the RNA–seq to genomic copies of L. polyedrum tandem repeat sequences was performed to assess the possibility of polycistronic transcripts, a hypothesis proposed to explain the lack of promoter elements in the intergenic region of the tandem repeat gene sequences. This analysis also showed a surprisingly high conservation of tandemly repeated gene sequences.   The transcriptome database was also used to fuel gene identification after protein sequencing by mass spectrometry, and a purified phosphoproteome fraction was found to be particularly amenable to high throughput approaches. A comparison of the phosphoproteome at two different times of day revealed that a major class of proteins whose phosphorylation state varied over time belonged to the RNA binding and translation GO category. The transcriptome was also used to define the spectrum of kinases present in L. polyedrum, which in turn was used to classify the different phosphorylated peptides as potential kinase targets. Predicted peptides of casein kinase 2 (CK2), a kinase known to be involved in the circadian clocks of other eukaryotes, were found to include many RNA binding proteins.  To assess the possibility that some of the many cold shock domain proteins identified in the transcriptome might modulate gene expression in L. polyedrum, as has been observed in many other eukaryotic and prokaryotic systems, the cellular response to cold temperatures was examined. Cold temperatures were found to induce rapid encystment, a metabolically inactive cell type whose role is to combat unfavourable environmental conditions. Changes in phosphoproteome profile were found to be the major molecular correlates to cyst formation. Predicted CK2 phosphosites are the most highly reduced class of kinase targets, a finding of interest as measurements of the bioluminescence rhythm confirmed that the clock is stopped in cysts

Keyphrases: Lingulodinium, RNA-Seq, Transcriptome, Transcription, Histones, Dinoflagellate, Lingulodinium, Gene Expression, Translation, Circadian Clock, Histones, Phosphoproteomics