Liu Dianyi, Wang Mingyu, Gent Jonathan I, Sun Peipei, Dawe R Kelly, Umen James
Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, Missouri, 63132, USA.
Institute of Bioinformatics, University of Georgia, Athens, Georgia, 30602, USA.
Plant J. 2025 Apr;122(2):e70153. doi: 10.1111/tpj.70153.
Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctate nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9-mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2. Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used cleavage under targets and tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5 kb (average lower estimate) to 175 kb (average upper estimate). The relatively small size of its centromeres suggests that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.
真核生物中的着丝粒由组蛋白H3变体CENP-A/CENH3的存在来定义。衣藻编码两种预测的CENH3旁系同源物，CENH3.1和CENH3.2，此前尚未对其进行表征。我们针对衣藻中两种预测的CENH3旁系同源物各自独特的N端表位生成了肽抗体，以及一种针对共享CENH3表位的抗体。所有三种CENH3抗体在免疫印迹上均识别出预期大小的蛋白质，并具有点状核免疫荧光染色模式。这些结果与两种旁系同源物均被表达并定位于着丝粒一致。利用CRISPR-Cas9介导的插入诱变在CENH3.1或CENH3.2中产生预测的无效突变。单突变体是可存活的，但cenh3.1 cenh3.2双突变体未获得，这证实了CENH3的功能是必不可少的。我们对来自CC-400和UL-1690菌株（CC-1690的衍生物）的两个染色体规模的衣藻基因组进行了测序和组装，分别有17条染色体中的17条和14条染色体具有完整的着丝粒序列，这使我们能够比较四个具有近乎完整组装的分离株之间的着丝粒进化。这些数据揭示了同源着丝粒在不同分离株之间的显著变化，包括构成衣藻主要着丝粒重复序列的ZeppL-LINE1（ZeppL）转座子的移动性和退化。我们使用靶点切割和转座酶标签化（CUT&Tag）来纯化和定位与CENH3结合的基因组序列，发现CENH3结合几乎完全富集在预测的着丝粒区域。一个有趣的例外是UL-1690中的2号染色体，其在遗传定位的着丝粒重复区域以及第二个远端位置有富集，该远端位置以一个最近获得的单一ZeppL插入为中心。衣藻17个着丝粒的CENH3结合区域范围从63.5 kb（平均下限估计）到175 kb（平均上限估计）。其着丝粒相对较小的尺寸表明衣藻可能是用于测试和应用人工染色体技术的有用生物体。
