Genetic variation in the anaphase promoting complex modulates fertility and chromosome alignment defects in the absence of KIF18A

The kinesin family member 18A (Kif18a) is an essential regulator of microtubule dynamics and chromosome alignment during mitosis. Laboratory mice with a loss of function mutation in Kif18a are are infertile due to mitotic arrest of germ cells, but this phenotype shows variable penetrance depending on genetic background. Kif18a is also necessary for mitotic progression of chromosomally unstable cancer cells, which exhibit mitotic arrest in the absence of KIF18A function. While it is clear that functional dependency on KIF18A varies by cell type and genetic context, the heritable factors that influence this dependency remain unknown. To address this, we took advantage of the variable penetrance observed in different mouse strain backgrounds to screen for genetic loci that modulate germ cell depletion in the absence of KIF18A. We found a significant association between the severity of germ cell depletion and a locus on Chr5 wherein anaphase promoting complex subunits 5 (Anapc5) and 7 (Anapc7) were the top candidate genes. We compared the expression of both genes at key timepoints in gondal development and found that both genes were differentially expressed in sensitive strain backgrounds when compared to resistant strain backgrounds. We also identify a novel retroviral insertion in Anapc7 that may in part explain the observed expression differences. In cell line models, we found that depletion of KIF18A induced mitotic arrest, which was partially rescued by co-depletion of APC7 and exacerbated by co-depletion of APC5. These findings suggest that differential expression and activity of Anapc5 and Anapc7 may influence sensitivity to KIF18A depletion in germ cells and CIN cells, with potential implications for optimizing antineoplastic therapies. We generated RNASeq data from testes and ovaries to evaluate the developmental and tissue specific expression profiles of the genes in the Chr5 QTL. Five biological replicates were collected from each sex (male, female), strain (C57BL/6J, CAST/EiJ, and B6CastF1), developmental timepoint~sex (ovaries at 12.5-13.5 days post coitum, testes at 5 days post partum). These developmental timepoints were chosen because mitotically dividing germ cells are enriched. In GEO metadata strain is designated under "genotype" as B6 (C57BL/6J: RRID:IMSR_JAX:000664), CAST (CAST/EiJ: RRID:IMSR_JAX:000928), and F1 offspring 'B6Cast' from a cross between B6 female x CAST male (C57BL/6J x CAST/EiJ F1).驱动蛋白家族成员18A（Kinesin Family Member 18A, Kif18a）是有丝分裂过程中调控微管动力学与染色体列队的关键调控因子。携带Kif18a功能丧失突变的实验小鼠会因生殖细胞有丝分裂阻滞而不育，但该表型的外显率会因遗传背景不同而存在差异。Kif18a同时也是染色体不稳定癌细胞完成有丝分裂进程所必需的，这类细胞在KIF18A功能缺失时会出现有丝分裂阻滞。尽管目前已明确，细胞类型与遗传背景会影响细胞对KIF18A的功能依赖性，但调控该依赖性的遗传因子仍未明确。为解决这一问题，本研究利用不同小鼠品系背景下观察到的可变外显率表型，筛选可调控KIF18A缺失时生殖细胞耗竭的遗传位点。我们发现生殖细胞耗竭的严重程度与5号染色体上的一个位点存在显著关联，该位点的候选核心基因为后期促进复合物亚基5（Anaphase Promoting Complex Subunits 5, Anapc5）与亚基7（Anaphase Promoting Complex Subunits 7, Anapc7）。我们检测了性腺发育关键时间点中这两个基因的表达水平，结果显示，相较于抗性品系背景，敏感品系背景下二者的表达均存在显著差异。我们还在Anapc7中发现了一处全新的逆转录病毒插入序列，这或许可以部分解释观测到的表达差异。在细胞系模型中，我们发现敲除KIF18A会诱导有丝分裂阻滞，而共敲除APC7可部分逆转该表型，共敲除APC5则会加剧阻滞现象。上述研究结果表明，Anapc5与Anapc7的差异表达与活性或许会影响生殖细胞与染色体不稳定癌细胞对KIF18A缺失的敏感性，这为优化抗肿瘤治疗方案提供了潜在思路。我们从睾丸与卵巢中获取了RNA测序（RNA-seq）数据，以分析5号染色体数量性状位点（Quantitative Trait Locus, QTL）内基因的发育与组织特异性表达谱。实验设置了两种性别（雄性、雌性）、三种品系（C57BL/6J、CAST/EiJ及B6CastF1）的生物学重复各5例，检测的发育时间点包括：交配后12.5~13.5天的卵巢，以及产后5天的睾丸。选择这些时间点是因为此时增殖的有丝分裂生殖细胞富集度较高。在GEO元数据中，品系信息以"genotype"字段标注：B6对应C57BL/6J（RRID:IMSR_JAX:000664），CAST对应CAST/EiJ（RRID:IMSR_JAX:000928），F1后代"B6Cast"则来自B6雌性×CAST雄性的杂交后代（C57BL/6J × CAST/EiJ F1）。