Preventing CpG hypermethylation in oocytes safeguards mouse development [Cut&Run]. Preventing CpG hypermethylation in oocytes safeguards mouse development [Cut&Run]

DNA methylation (DNAme) controls gene expression, genome stability and cell identity in many mammalian organisms. Genomes of most somatic cells and spermatozoa are largely methylated, apart from CpG-dense sequences including gene promoters1. In mouse oocytes, DNAme is mostly limited to transcribed regions2,3. The mechanisms restricting global DNAme acquisition in developing oocytes and the relevance thereof for regulating gene expression and embryo development after fertilization are unknown. Here we show that the histone H3 lysine 36 dimethyl (H3K36me2) demethylases KDM2A and KDM2B redundantly execute multiple chromatin functions during oocyte development, which are vital to pre- and post-implantation development. Firstly, by serving as recruitment factors of variant Polycomb Repressive Complex 1 (vPRC1), they control genome-wide H2A mono-ubiquitination deposition (H2AK119u1) and PRC1-dependent gene repression4. Secondly, as demethylases, KDM2A/KDM2B prevent global H3K36me2 accumulation, thereby impeding DNMT3A-catalyzed de novo DNAme within PRC1-controlled promoter, genic, and intergenic regions, and even at non-PRC1-controlled CpG-dense gene promoters5. Decisively, we demonstrate that aberrant Dnmt3a-dependent DNAme established in Kdm2a/Kdm2b double mutant oocytes represses transcription from maternal loci in two-cell embryos and impairs pre-implantation development. Hence, KDM2A/KDM2B are essential for defining the appropriate oocyte DNA methylome which in turn conveys competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient to erase aberrant DNAme from maternal chromatin. Lastly, our work shows that early development is vulnerable to gene dosage haplo-insufficiency effects, possibly in a parental-specific manner. Overall design: To study the function of KDM2A and KDM2B in regulating genome wide H2AK119u1, H3K36me2 and H3K36me3 in oocytes, we conditionally altered their expression in growing oocytes in several ways. First, by using Zp3-driven CRE recombinase-mediated excision, we removed exons encoding the histone demethylase JmjC domains of Kdm2a or Kdm2b, which fully abrogated expression of either protein. We also generated mice expressing a KDM2B protein lacking its CxxC domain, unable to be recruited to CpG island regions of the mouse genome. To address possible combinatorial roles of both proteins, we generated oocytes conditionally deficient for Kdm2a in combination with either Kdm2b mutation. We profiled genome-wide histone modification marks (H2AK119u1, H3K36me2 and H3K36me3) by Cut &Run method in fully grown germinal vesicle oocytes of following genotypes: (1) Kdm2a-KO; Kdm2b-deltaCxxC, (2) Kdm2a-KO; Kdm2b-KO and (3) controls.

DNA甲基化（DNA methylation, DNAme）在众多哺乳动物体内调控基因表达、维持基因组稳定性并决定细胞身份。多数体细胞与精子的基因组整体呈现高水平甲基化，仅除开包含基因启动子在内的CpG密集序列区域¹。而在小鼠卵母细胞中，DNA甲基化则主要局限于转录区域²,³。目前，调控发育中卵母细胞全基因组DNA甲基化建立的分子机制，以及该过程对受精后基因表达与胚胎发育的调控意义仍不明确。本研究发现，组蛋白H3赖氨酸36二甲基化（H3K36me2）去甲基化酶KDM2A与KDM2B在卵母细胞发育过程中冗余发挥多种染色质调控功能，这对胚胎植入前与植入后发育均至关重要。其一，作为多梳抑制复合体1变体（variant Polycomb Repressive Complex 1, vPRC1）的招募因子，二者可调控全基因组范围的组蛋白H2A单泛素化沉积（H2AK119u1）以及依赖PRC1的基因沉默⁴。其二，作为去甲基化酶，KDM2A/KDM2B可阻止全基因组H3K36me2的积累，从而阻碍DNA甲基转移酶3A（DNMT3A）催化的从头DNA甲基化（de novo DNAme）在PRC1调控的启动子、基因区以及基因间区发生，甚至可作用于非PRC1调控的CpG密集型基因启动子⁵。关键而言，本研究证实，在Kdm2a/Kdm2b双突变卵母细胞中建立的、依赖Dnmt3a的异常DNA甲基化，会抑制二细胞胚胎中母源基因座的转录，并损害胚胎植入前发育。因此，KDM2A/KDM2B对于塑造正常的卵母细胞DNA甲基化组至关重要，而该甲基化组可赋予早期胚胎发育潜能。本研究提示，早期胚胎所具备的卓越重编程能力，不足以清除母源染色质中的异常DNA甲基化。最后，本研究表明早期发育易受基因剂量单倍体不足效应的影响，且该效应可能呈现亲本特异性。实验整体设计：为研究KDM2A与KDM2B在卵母细胞中调控全基因组H2AK119u1、H3K36me2与H3K36me3修饰的功能，我们通过多种方式对生长中卵母细胞内二者的表达进行条件性调控。其一，利用Zp3驱动的CRE重组酶介导的基因敲除，我们剔除了Kdm2a或Kdm2b中编码组蛋白去甲基化酶JmjC结构域的外显子，从而完全阻断对应蛋白的表达。此外，我们构建了表达缺失CxxC结构域的KDM2B蛋白的小鼠，该突变蛋白无法被招募至小鼠基因组的CpG岛区域。为探究二者可能存在的协同调控功能，我们构建了同时条件性缺失Kdm2a并携带Kdm2b突变的卵母细胞。我们通过Cut&Run技术，对以下基因型的完全生长型生发泡卵母细胞（germinal vesicle oocytes）的全基因组组蛋白修饰标记（H2AK119u1、H3K36me2与H3K36me3）进行了检测：(1) Kdm2a-KO; Kdm2b-ΔCxxC，(2) Kdm2a-KO; Kdm2b-KO，以及(3) 野生型对照。