The key role of CYC2 during meiosis in Tetrahymena thermophila

Abstract

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.

Keywords: RNA-Seq; Tetrahymena thermophila; cyclin; homologous recombination; meiosis.

Figure 1

Transcriptional expression profile of CYC2 (TTHERM_00079530) in T. thermophila and confirmation of CYC2 knock-out strains. (A) The line chart was generated with microarray data of mRNA products of all time points (three GROWTH time points, seven STARVATION time points and ten conjugation time points) in life cycle of T. thermophila. The solid and dashed lines denoted the expression values normalized by two different methods. For vegetatively growing cells, L-l, L-m, and L-h respectively correspond to ~1 × 10⁵ cells/mL, ~3.5 × 10⁵ cells/mL, and ~1 × 10⁶ cells/mL. Samples collected at 0, 3, 6, 9, 12, 15, and 24 h after starvation began were respectively referred to as S-0, S-3, S-6, S-9, S-12, S-15, and S-24, and at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h after mixing equal volumes of B2086 and CU428 cells for conjugation initiation referred to as C-0, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16, and C-18. It is manifested that CYC2 expressed at transcriptional level exclusively during conjugation and abundantly during 2–4 h after conjugation initiation followed by additional expression around 12–14 h. (B) The CYC2 knock-out strains (ΔCYC2) of two different mating types (B2086 and Cu428) were confirmed by whole cell extract PCR. The difference of PCR products lengths between strains of two mating types (marked as KO-1 and KO-2) was reasonable for a deleted region larger than target sequence, and it was often observed during the process of co-deletion (Hayashi and Mochizuki, 2015). (C) The fold changes of mRNA expression level of paired strains of ΔCYC2 at 4 time points after conjugation initiation were examined by RT-qPCR using two different primer sets in CDS (‘qPCR mRNA check-1’ primers for RT-qPCR 1 and ‘qPCR mRNA check-2 exon1’ primers for RT-qPCR 2) (Fig. S1). N number of biological repeats

Figure 2

Phenotypic analysis of ΔCYC2. (A) The time course analysis of the progression of the nuclear events showed that the loss of CYC2 resulted in an early conjugation arrest where the conjugants were not able to pass through the crescent stage successfully and lack of meiosis division. The conjugating process was artificially separated into seven parts according to the morphology characteristics (unpaired, pair formed, crescent, meiosis divisions, pro-nuclear exchange, post-zygotic mitosis, and exconjugant) and the related graphical representation were highlighted in different color at the right side. The 4 independent biological repeats of cell samples were collected respectively at 2, 3, 4, 6, 10, and 24 h after conjugation initiation. At least 100 pairs of cells were counted in each repeat. The percentage of each morphologic status was calculated and displayed in the histographs above. (B) The status of cell cycle arrest of ΔCYC2 before crescent stage was captured during microscopic observation of cells stained with DAPI at 3 h when crescent shape formed in wild type conjugants and at 4.5 h when meiosis II was finished

Figure 3

Overview of differentially expressed genes (DEGs). (A) DEG number of CYC2 KO strains and wild type strains at four time stages (2, 2.5, 3, and 3.5 h after conjugation initiation). (B) Venn diagram identifying transcriptome DEG features between KO strains and wild type strains at four different time stages

Figure 4

Gene ontology (GO) enrichment analysis of down regulated genes. The filtered significantly down regulated genes (Log2 (fold change) < −2 and P value < 0.05) of each time point were subjected to GO enrichment analysis separately in biological process, molecular biology, and cellular function. (A) Percentage of associated genes/term (%) at 2 h. (B) Percentage of associated genes/term (%) at 2.5 h. (C) Percentage of associated genes/term (%) at 3 h. (D) Percentage of associated genes/term (%) at 3.5 h. The length of bars from each histogram indicates the percentage of associated genes for each term. The number on top of each bar means the number of associated genes

Figure 5

Immunostaining of wild type and CYC2 KO strains. (A) Gamma-H2AX signals occupied the whole wild type micronuclei before crescent stage. (B) Gamma-H2AX signals occupied the whole wild type meiotic micronuclei during crescent stage. (C and D) There were no gamma-H2AX signals in CYC2 KO micronuclei, which could not elongate fully during conjugation

Figure 6

RT-qPCR verification of the RNA-Seq results. The 8 randomly selected genes (TTHERM_00961910; TTHERM_00730300; TTHERM_00426250; TTHERM_00077290; TTHERM_00865200; TTHERM_00338290; TTHERM_01109940; TTHERM_00763040) plus CYC2 TTHERM_00079530) and SPO11 (TTHERM_00627090) were performed with RT-qPCR. The primers were designed to across an intron to eliminate potential confluence from residue genomic DNA contamination. cDNA templates were from of 3 independent biological repeats of cell samples respectively at 2 h (A), 2.5 h (B), 3 h (C), and 3.5 h (D) after conjugation initiation. The tendencies of mRNA expression changes were in accordance with the RNA-Seq analysis results, which well-validated of the sequencing data