Autophagy regulates early embryonic development in mice via affecting H3K4me3 modification

doi:10.12307/2026.022

Abstract

Abstract: BACKGROUND: Autophagy, as a key regulatory mechanism of cell development, plays an important role in different stages of embryonic development. The mechanism of how autophagy regulates embryonic development through histone modifications is currently unclear.
OBJECTIVE: To investigate the effect of autophagy on trimethylation of lysine 4 on histone H3 (H3K4me3) modification in embryos and its effect on embryonic development.
METHODS: Mouse fertilized eggs were divided into control and autophagy inhibitor-treated groups (chloroquine phosphate-treated group and 3-methyladenine-treated group), and cultured in vitro to different periods of time, and were then classified as early 2-cell embryos, middle 2-cell embryos, late 2-cell embryos, 4-cell embryos, 8-cell embryos, morula stage, and blastocyst stage. Levels of reactive oxygen species, autophagy marker proteins LC3B and P62, DNA loss marker γH2AX, and H3K4me3 were analyzed by immunofluorescence assay in late 2-cell embryos of each group. Changes in H3K4me3 modification in late 2-cell embryos of each group were detected by CUT&Tag.
RESULTS AND CONCLUSION: (1) Autophagy inhibition caused embryo development arrest. (2) There was no significant difference in reactive oxygen species and γH2AX between the autophagy inhibitor-treated groups and control group. (3) H3K4me3 levels were significantly elevated in the autophagy inhibitor-treated group compared with the control group. (4) CUT&Tag results showed a significantly increased H3K4me3 peaks on the proximal promoter region of the genes after autophagy inhibition and an increase of H3K4me3-specific modification genes. These findings suggest that autophagy may affect embryonic development by regulating the level of H3K4me3 modification.

Key words: mouse, autophagy, H3K4me3, embryonic development, histone modification, reactive oxygen species, DNA damage, mouse embryo, methylation

CLC Number:

Hu Jing, Zhu Ling, Xie Juan, Kong Deying, Liu Doudou. Autophagy regulates early embryonic development in mice via affecting H3K4me3 modification[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(5): 1147-1155.

Figures/Tables 5

2.2 自噬抑制不诱导胚胎的活性氧和DNA损伤 3-MA和磷酸氯喹处理后检测小鼠晚期2-细胞胚胎的活性氧和DNA损伤标记物γH2AX，结果显示，3-MA处理组(n=18)和磷酸氯喹处理组(n=17)的活性氧水平与对照组(n=15)无明显差异(P > 0.05)，见图2A，B。γH2AX的免疫染色结果表明，3-MA处理组(n=19)与对照组(n=16)相比没有显著差异(P > 0.05)，磷酸氯喹处理组(n=13)相对于对照组(n=16)没有显著差异(P > 0.05)，见图2C，D。综合以上结果表明自噬抑制对胚胎发育的影响不是由活性氧和DNA损伤引起的。 2.3 自噬抑制导致胚胎H3K4me3修饰水平增强 2.3.1 各阶段胚胎的H3K4me3水平为了明确小鼠早期胚胎发育过程中H3K4me3修饰的动态变化，通过免疫荧光检测了小鼠胚胎从受精卵到囊胚阶段的H3K4me3水平。结果显示，H3K4me3修饰水平从受精卵到早期2-细胞阶段增强，从中期2-细胞到4-细胞阶段逐渐减弱并降到最低；从4-细胞一直到囊胚阶段又逐渐升高，但这一阶段H3K4me3的总体表达量相对于合子到中期2-细胞阶段较低，见图3A，B。 2.3.2 自噬抑制后H3K4me3的表达为了明确自噬抑制后对H3K4me3修饰是否产生影响，用3-MA和磷酸氯喹处理胚胎后通过免疫荧光检测了晚期2-细胞胚胎的H3K4me3的表达量。结果显示，3-MA(n=9)和磷酸氯喹处理组(n=17)的H3K4me3水平相较于对照组(n=17)均显著升高(P < 0.001)，见图3C，D。以上结果表明，H3K4me3在胚胎发育过程中经历了修饰逐渐减少然后又逐渐增多的动态变化，在晚期2-细胞时期，胚胎的H3K4me3修饰水平降到了较低水平，自噬抑制剂导致该时期胚胎的H3K4me3修饰水平升高，这可能是自噬抑制造成胚胎发育阻滞的原因之一。 2.4 自噬抑制导致胚胎的H3K4me3修饰模式和强度改变为进一步确定自噬抑制对H3K4me3修饰的影响，进行了CUT&Tag检测分析。结果显示，相较于对照组，磷酸氯喹处理组H3K4me3整体的修饰水平增加(图4A)。通过分类H3K4me3修饰区域，结果显示磷酸氯喹处理后，H3K4me3在近端启动子区域内富集明显增加(图4B)，且热图分析和IGV示例图显示，H3K4me3在转录起始位点(TSS)附近修饰增强(图4C，D)。对H3K4me3修饰基因进行分类分析，结果显示磷酸氯喹处理后，H3K4me3修饰水平特异性增加的基因数量显著增多(图4E)，对修饰水平特异性增加的基因进行GO功能富集分析显示，这些基因主要参与细胞形态发生、细胞连接和细胞增殖的调控(图4F)。以上结果说明自噬通过改变H3K4me3的修饰模式，影响与细胞形态、细胞连接和细胞增殖等关键生物学过程，从而调控胚胎的正常发育。"

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