Tet3-mediated demethylation is required for dorso-ventral cone opsin patterning and proper specification of cone and bipolar cells in mice

Abstract Active demethylation of genomic DNA in mammals is initiated by the enzymatic activity of TET1, TET2, or TET3, members of the TET enzyme family, which are αKG-dependent 5-methylcytosine (5mC) dioxygenases that oxidatize 5mC to 5-hydoxymethylcytosine (5hmC), 5-formyl- (5fC) and 5-carboxycytosine (5caC). Among the three TET family members, TET3 shows the highest expression in terminally differentiated neurons. Pathogenic variants in TET3 cause a rare autosomal dominant neurodevelopmental disorder termed Beck-Fahrner syndrome, characterized by intellectual disability and global developmental delay, autistic traits and behavioral abnormalities, and varying degrees of growth abnormalities (both over- and undergrowth), hypotonia, movement disorders, seizures and distinctive facial dysmorphism. Approximately half of patients exhibit various ophthalmological findings, such as nystagmus (involuntary eye movements), strabismus, or refractive errors, but thorough clinical description of the ocular manifestation is still lacking. Thus, visual impairment may be a significant but previously underrecognized component of the clinical phenotype of patients with TET3 deficiency. Here, we investigated the functional role of Tet3 in the retina using conditional Tet3 mutant mice lacking Tet3 in all retinal cells. Tet3-deficient mice showed an increase in the number of cone photoreceptors and a decrease in bipolar cells. At the functional level, Tet3-deficient mice presented with reduced light responses in photopic electroretinography measurements. Absence of Tet3 function lead to a decrease in genomic 5hmC content and loss of 5fC in the retina. Tet3-deficiency induced transcriptional and proteome changes consistent with a key role of Tet3-mediated demethylation during retinal development. Together, these findings highlight the role of Tet3 in in retinal cell type specification and retinal function.摘要 哺乳动物基因组DNA的主动去甲基化由TET酶家族（TET enzyme family）成员TET1、TET2或TET3的酶活性启动，该家族成员为αKG依赖型5-甲基胞嘧啶（5-methylcytosine, 5mC）双加氧酶，可将5mC氧化为5-羟甲基胞嘧啶（5-hydoxymethylcytosine, 5hmC）、5-甲酰胞嘧啶（5-formylcytosine, 5fC）与5-羧基胞嘧啶（5-carboxycytosine, 5caC）。在TET家族的三个成员中，TET3在终末分化神经元中的表达水平最高。TET3的致病变异会引发一种罕见的常染色体显性神经发育障碍，即Beck-Fahrner综合征（Beck-Fahrner syndrome），其临床表现为智力障碍、全面发育迟缓、自闭症特征与行为异常，以及不同程度的生长异常（过度生长与生长不足）、肌张力低下、运动障碍、癫痫发作及特征性面部畸形。约半数患者会出现多种眼部异常，如眼球震颤（nystagmus）、斜视（strabismus）或屈光不正，但目前仍缺乏对眼部表型的全面临床描述。因此，视力损伤可能是TET3缺陷患者临床表型中一个重要但此前未被充分认识的组成部分。本研究针对所有视网膜细胞均缺失Tet3的条件性Tet3突变小鼠，探究了Tet3在视网膜中的功能作用。结果显示，Tet3缺陷小鼠的视锥光感受器细胞数量增加，双极细胞数量减少。在功能层面，Tet3缺陷小鼠的明视视网膜电图（photopic electroretinography）检测显示其光反应减弱。Tet3功能缺失会导致视网膜中基因组5hmC含量降低，并造成5fC丢失。Tet3缺陷引发的转录组与蛋白质组变化，与Tet3介导的去甲基化在视网膜发育过程中的关键作用相符。综上，这些研究结果凸显了Tet3在视网膜细胞类型特化及视网膜功能维持中的作用。