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 before 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 before 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 gene ontology 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.

FIGURE 1:

Validation of cell cycle synchrony by centrifugal elutriation. (A) Cells isolated at 30 min intervals after elutriation were stained with propidium iodide (PI) and analyzed by flow cytometry (adjacent lanes correspond to biological replicates). (B) EdU incorporation into micro- and macronuclei following centrifugal elutriation as determined by fluorescence microscopy of fixed cells. (C) Summary timeline for macro- and micronuclear cell cycle progression. (D) Pipeline of sample processing for RNA-seq and cytological landmark determination. (E) Heatmap of cell cycle–regulated genes. Periodically expressed genes identified by MetaCycle for genes having 1.5-fold changes in two opposite directions.

FIGURE 2:

Cell cycle–regulated histones and chromatin modifier genes. (A) Expression profile of core histone genes. (B) Expression profile of macronuclear-specific histone variants. (C) Expression profile of the micronuclear-specific, centromere-associated histone H3 variant, CNA1. (D) Expression profiles of macro- and micronuclear-specific H1 histones (dashed line: MAC specific; dotted line: MIC specific). (E) Cell cycle–regulated histone modifiers (see the text for gene names).

FIGURE 3:

Expression profile of cohensin and condensing proteins. (A) Expression profile of cell cycle–regulated condensin genes (dashed lines: macronuclear specific; dotted lines: micronuclear specific; solid line: present in both nuclei). (B) Expression profile of cell cycle–regulated cohesin genes (all micronuclear specific).

FIGURE 4:

Expression profile of macro- or micronuclear-specific nucleoporin genes. (A) Cell cycle–regulated nucleoporins (dotted lines: micronuclear specific, solid lines: present in both nuclei). (B) Cell cycle–regulated expression of macronuclear- and nonnuclear-specific importin-α genes; IMA1 (macronuclear specific), IMA6, and IMA9. (C) Cell cycle–regulated expression of micronuclear importin-α genes. (D) Phylogenetic tree analysis of importin-α genes. (E) Cell cycle–regulated β-like importin proteins.

FIGURE 5:

Oscillating cyclin, CDK, and E2F mRNA abundance profiles. (A) Cell cycle–regulated expression of cyclin D gene family members. (B) Cell cycle–regulated expression of cyclin A/B genes. Solid lines: pattern 1 for cyclin A/B gene family members; dotted lines: pattern 2 for cyclin A/B gene family members. (C) Cell cycle–regulated CDK genes. (D) Cell cycle–regulated E2Fs. Solid line: potential E2F activator; dotted lines: potential E2F repressors and atypical E2F with two DNA-binding domains.

FIGURE 6:

Cluster and GO enrichment analysis. (A) Clusters of seven distinct periodically gene expression profiles and four subclusters (5A–D). (B) Overrepresented GO biological processes of four clusters. Overrepresented GO biological processes of subclusters within cluster 5. (C) Gene expression profiles of DNA replication protein-coding genes within cluster 7. Gene expression profile of mitotic genes within cluster 1. Gene expression profile of mitotic genes of subcluster 5C. Gene expression profile of mitotic and cell cycle checkpoint genes of subcluster 5D. (D) Coregulation analysis of the MCM6 mRNA cell cycle profile: top 10 coregulated genes.