Characterization of the enzyme for 5-hydroxymethyluridine production and its role in silencing transposable elements in dinoflagellates. Characterization of the enzyme for 5-hydroxymethyluridine production and its role in silencing transposable elements in dinoflagellates

Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homologue, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae, we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements (TEs), implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark. Overall design: In our study, we successfully obtained the near chromosome-scale genome assembly of A. carterae based on a combination of genome survey approach, long-read sequencing through Pacbio sequel II platform with the circular consensus sequencing (CCS) mode, and Hi-C sequencing data. For A. carterae genome annotation, we also performed transcriptome sequencing using both short-read RNA-seq (DNBSEQ PE150) and long-read sequencing (PacBio Sequel platform). To map the genome-wide distribution of 5hmU sites in A. carterae, we utilized an anti-5hmU antibody-based DNA immunoprecipitation method. To examine the effects of altered 5hmU levels on repeat elements, we treated A. carterae cells with and without 5hmU synthase inhibitor, 2,4-pyridinedicarboxylic acid (2-4P), followed by directional RNA sequencing to reveal their transcriptional change. Additionally, we conducted Whole Genome Bisulfite Sequencing (WGBS) to investigate the correlation between 5hmU and genome-wide 5mC loci in A. carterae. All treatments in this study have at least two biological replicates.

甲藻（Dinoflagellate）的染色体极具特殊性：其染色体组装不依赖于典型真核生物所必需的结构性核小体，且呈现胆甾型液晶态。5-羟甲基尿苷（5-hydroxymethyluridine，5hmU）在甲藻染色体DNA中的含量异乎寻常地高，其功能数十年来始终是一个未解之谜。本研究证实，不同甲藻的5hmU修饰水平存在差异，且该修饰通过胸腺嘧啶羟化途径生成。重要的是，我们通过体内与体外生化实验，鉴定出了催化5hmU产生的酶——一种推定的甲藻TET/JBP同源蛋白。基于卡特前沟藻（Amphidinium carterae）的近染色体水平基因组组装结果，我们绘制了全面的5hmU修饰图谱，发现5hmU位点在重复序列元件中显著富集。此外，利用双加氧酶抑制剂抑制5hmU修饰后，携带5hmU标记的转座因子（transposable elements，TEs）的转录水平被激活，这表明5hmU可能作为表观遗传标记参与转座子沉默过程。综上，本研究揭示了甲藻5hmU的生物合成途径、全基因组分布特征与分子功能，为解析这一神秘DNA修饰的功能提供了机制层面的见解。

整体实验设计：本研究结合基因组勘测分析、PacBio Sequel II平台环形一致测序（circular consensus sequencing，CCS）模式的长读长测序数据与Hi-C测序数据，成功获得了卡特前沟藻的染色体级基因组组装结果。在卡特前沟藻基因组注释环节，我们同时采用短读长RNA测序（DNBSEQ PE150）与长读长测序（PacBio Sequel平台）开展了转录组测序。为绘制卡特前沟藻全基因组范围内的5hmU位点分布，我们利用基于抗5hmU抗体的DNA免疫沉淀技术。为探究5hmU水平改变对重复序列元件的影响，我们分别用5hmU合酶抑制剂2,4-吡啶二羧酸（2,4-pyridinedicarboxylic acid，2-4P）处理卡特前沟藻细胞，并设置未处理对照组，随后通过定向RNA测序分析两组的转录组变化。此外，我们开展了全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing，WGBS），以探究卡特前沟藻中5hmU与全基因组5mC位点之间的关联。本研究中的所有处理组均设置至少2次生物学重复。