Transcriptome analysis of the binucleate ciliate Tetrahymena thermophila with asynchronous nuclear cell cycles

Abstract

Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the ‘cell cycles’ of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells prior to onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed prior to their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER GO terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication and cell division.

Revealing Gene Expression Heterogeneity in a Clonal Population ofTetrahymena thermophilathrough Single-Cell RNA Sequencing

“…Among the nine genes analyzed, one was designated as encoding hypothetical proteins, three lacked GO terms and the remaining five were identified as the genes associated with intracellular anatomical structure, translation, protein folding, membrane transport and electron transfer. To observe gene expression patterns of cells in each cell cycle, we scored cells based on the genes that had been previously reported as cell cycle-dependent (Zhang et al, 2023) and mapped on UMAP (Fig. 3 (a)).…”

“…Seurat. The markers of each cell phase (Table S2) are based on the list from Zhang et al (2023). This score was then used to regress out the effect of the cell phase.…”

“…A primary contributing factor for the expression variability obtained from the scRNA-seq has been proposed to be cell cycle phase. To remove the variations from cell cycle phases and reveal heterogeneity from other sources, we regressed out the contribution from cell cycle phase by utilizing a recently reported set of genes with cell-cycle-dependent expression patterns (Zhang et al, 2023).…”

Developmentally Programmed Switches in DNA Replication: Gene Amplification and Genome-Wide Endoreplication in Tetrahymena

“…Like all ciliated protozoa, T. thermophila harbors two functionally distinct nuclei within its cytoplasm: the transcriptionally silent, diploid ‘germline’ micronucleus and the transcriptionally active, polyploid ‘somatic’ macronucleus (reviewed in [ 11 ]). ORC-dependent DNA replication is coordinately regulated in vegetative growing Tetrahymena , such that the heterochromatic micronucleus and euchromatic macronucleus replicate their chromosomes at non-overlapping stages of the cell cycle (reviewed in [ 12 , 13 ]). Development is much more complex: multiple rounds of micro- and macronuclear DNA replication, meiosis, and mitosis occur in parental gametes and post-zygotic progeny.…”

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

“…While HHT3 (H3.3) is constitutively expressed in growing and starved cells [40,41], HHT4 (H3.4, not to be confused with testis-specific H3.4 found in animals) is very weakly expressed if at all [39]. A recent study has shown that the HHT2 expression level peaks in S phase, while the HHT3 level remains stable across the cell cycle in Tetrahymena [42]. Although, canonical H3s are strictly deposited in an RD manner, H3.3 and H3.4 can be deposited both by a transcriptionassociated RI pathway and inefficiently by an RD pathway [39].…”