Binding of zebrafish lipovitellin and L1‑ORF2 increases the accessibility of L1‑ORF2 via interference with histone wrapping

Abstract

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 in vitro 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 in vitro 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.