Department of Genetics, Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China.
Department of Genetics, Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China.
Int J Mol Med. 2025 Jan;55(1). doi: 10.3892/ijmm.2024.5443. Epub 2024 Oct 25.
Long interspersed nuclear element‑1 (L1) is highly expressed in the early embryos of humans, rodents and fish. To investigate the molecular mechanisms underlying high expression of L1 during early embryonic development, a C1‑open reading frame (ORF)2 vector was constructed in which ORF2 of human L1 (L1‑ORF2) was inserted into a pEGFP‑C1 plasmid. C1‑ORF2 vector was injected into early zebrafish embryos (EZEs) to observe expression of EGFP reporter protein by fluorescence microscopy. RNA‑seq and RT‑qPCR were used to detect the effects of lipovitellin (LV) on gene expression in EZEs. The binding ability of LV to L1‑ORF2 DNA was detected by electrophoretic mobility‑shift assay (EMSA). The chromatin recombinant DNase I digestion and ATAC‑seq assay were used to evaluate the accessibility of plasmid DNA. C1‑ORF2 vector induced high expression of enhanced green fluorescent protein (EGFP) reporter gene after it had been injected into 0 h post‑fertilization (hpf) zebrafish embryos, although histone octamer inhibited expression of EGFP in C1‑ORF2. SDS‑PAGE was used to show that LV was the predominant protein binding ORF2 DNA in 0 hpf zebrafish embryo lysate (ZEL). Both ZEL and purified LV from ZEL attenuated the inhibitory effects induced by histone. LV bound histone to interfere with the binding of histone to ORF2 DNA. Both chromatin reconstitution experiments and assay for transposase‑accessible chromatin with sequencing with HeLa cells were utilized to demonstrate that the interference induced by LV resulted in increased accessibility of C1‑ORF2. Transcription experiments verified that LV could enhance the mRNA levels of zebrafish early embryo expression genes grainyhead‑like transcription factor 3 (GRHL3), SRY‑box transcription factor 19a (SOX19A) and nanor (NNR) and also of the EGFP gene. LV was found to increase the expression levels of the zebrafish early embryo expression genes in liver tissue after LV had been injected into the abdominal cavity of adult male zebrafish. Taken together, the findings of the present study demonstrated that LV activates the expression of EGFP induced by ORF2 in EZEs by enhancing the accessibility of ORF2 DNA.
长散在核元件 1（L1）在人类、啮齿动物和鱼类的早期胚胎中高度表达。为了研究 L1 在早期胚胎发育过程中高表达的分子机制，构建了一个 C1-开放阅读框（ORF）2 载体，其中人类 L1 的 ORF2（L1-ORF2）插入到 pEGFP-C1 质粒中。将 C1-ORF2 载体注射到早期斑马鱼胚胎（EZEs）中，通过荧光显微镜观察 EGFP 报告蛋白的表达。使用 RNA-seq 和 RT-qPCR 检测卵黄蛋白（LV）对 EZEs 基因表达的影响。通过电泳迁移率变动分析（EMSA）检测 LV 与 L1-ORF2 DNA 的结合能力。使用染色质重组 DNA 酶 I 消化和 ATAC-seq 分析评估质粒 DNA 的可及性。将 C1-ORF2 载体注射到受精后 0 小时（hpf）的斑马鱼胚胎中，诱导增强型绿色荧光蛋白（EGFP）报告基因的高表达，尽管组蛋白八聚体抑制了 C1-ORF2 中的 EGFP 表达。SDS-PAGE 显示，LV 是 0 hpf 斑马鱼胚胎裂解液（ZEL）中 ORF2 DNA 的主要结合蛋白。ZEL 和从 ZEL 中纯化的 LV 均减弱了组蛋白诱导的抑制作用。LV 结合组蛋白以干扰组蛋白与 ORF2 DNA 的结合。使用 HeLa 细胞进行染色质重建实验和转座酶可及染色质测序分析均表明，LV 诱导的干扰导致 C1-ORF2 的可及性增加。转录实验验证了 LV 可以增强斑马鱼早期胚胎表达基因颗粒头样转录因子 3（GRHL3）、SRY 盒转录因子 19a（SOX19A）和 nanor（NNR）以及 EGFP 基因的 mRNA 水平。向成年雄性斑马鱼的腹腔注射 LV 后，发现 LV 增加了早期胚胎表达基因在肝脏组织中的表达水平。综上所述，本研究结果表明，LV 通过增强 ORF2 DNA 的可及性，激活 EZEs 中 ORF2 诱导的 EGFP 表达。
