Cyclin Cyc2 is required for elongation of meiotic micronucleus in Tetrahymena thermophila

Meiosis occurs in all sexually reproducing unicellular and multicellular eukaryotes. Bouquet formation is indispensible for homologous pairing or recombination during meiotic prophase I, but the regulatory mechanism of this process remain largely unknown. Cyclins regulate the precise meiosis progression by activating cyclin-dependent kinases. We therefore investigated the functional contribution of cyclin Cyc2p during bouquet (named crescent in Tetrahymena) formation in Tetrahymena thermophila. As a conjugation specific gene, CYC2 expression is significantly upregulated at 2-4 h after the initiation of conjugation and Cyc2p mainly localized in cytoplasm as well as weakly in the meiotic micronucleus (Mic). CYC2 knockout mutants failed to form elongated crescent structure and aborted meiotic development. Mic DNA double-strand breakage (DSB) decreased in ?CYC2 cells as shown by ?-H2A.X staining, consistent with the finding that expression level of SPO11, DMC1, and RAD51 decreased in ?CYC2 cells. However, ?CYC2 cells failed to form crescent structure when artificial DSBs were induced, indicating that the inability to enter crescent phase was not completely due to the lack of DSBs. The localization of tubulin showed that impaired structure of nuclei and nuclear membrane may contribute to the blocked Mic elongation. This is further supported by the observation that expression levels of two microtubule associated kinesin genes, KIN11 and KIN141, were significantly downregulated in ?CYC2 cells. Interestingly, scnRNA accumulation seemed intact in ?CYC2 cells whereas the intensity of the heterochromatin marker H3K23me3 was abnormally increased. Together, these results showed that cyclin Cyc2p is required for micronuclear meiosis by controlling meiotic prophase chromosome breakage and the microtubule movement of nuclei in Tetrahymena. Overall design: Examination of transcriptional variation associated with deletion of CYC2 gene

减数分裂广泛存在于所有进行有性生殖的单细胞与多细胞真核生物中。花束结构形成（bouquet formation）是减数分裂前期I同源配对与重组的必要前提，但该过程的具体调控机制目前仍不明晰。细胞周期蛋白（cyclins）通过激活细胞周期蛋白依赖性激酶（cyclin-dependent kinases），精准调控减数分裂的进程。为此，我们以嗜热四膜虫（Tetrahymena thermophila）为研究模型，探究了细胞周期蛋白Cyc2p在花束结构（该结构在嗜热四膜虫中被称为新月形结构（crescent））形成过程中的功能作用。CYC2作为接合生殖特异性基因，其表达水平在接合生殖启动后的2~4小时内显著上调；Cyc2p主要定位于细胞质，同时在减数分裂小核（micronucleus, Mic）中存在微弱表达。CYC2基因敲除突变体无法形成伸长的新月形结构，减数分裂发育进程完全停滞。通过γ-H2A.X染色检测发现，ΔCYC2细胞中的小核DNA双链断裂（double-strand breakage, DSB）水平显著降低，这与ΔCYC2细胞中SPO11、DMC1及RAD51的表达水平下调的实验结果相一致。但当人工诱导产生DSB时，ΔCYC2细胞仍无法形成新月形结构，这表明细胞无法进入新月形阶段并非完全由DSB缺失所导致。微管蛋白定位实验结果显示，细胞核与核膜结构受损可能是小核伸长受阻的关键原因。ΔCYC2细胞中两个微管相关驱动蛋白基因KIN11与KIN141的表达水平显著下调，这一结果进一步佐证了上述结论。有趣的是，ΔCYC2细胞中的扫描小核RNA（scnRNA）积累水平似乎未受影响，但异染色质标记物H3K23me3的信号强度却异常升高。综上，本研究结果表明，在嗜热四膜虫中，细胞周期蛋白Cyc2p通过调控减数分裂前期染色体断裂与细胞核微管运动，对小核减数分裂过程至关重要。整体实验设计：探究与CYC2基因缺失相关的转录组差异。