Human Endogenous Retrovirus K (HERV-K) can drive gene expression as a promoter in Caenorhabditis elegans

Figures

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  Fig. 1. Promoter activity of HERV-K119 LTR in Caenorhabditis elegans. Merged DIC and fluorescence images are presented. Arrows indicate (A) neuronal precursors in embryos, (B) muscle cells and neurons in the head and tail in early L1 larva, (C) neurons in L2-L3 larva, (D) intestine of L4 larva, and (E) vulva in adults. (F) Selective GFP expression in the vulva muscles of adult worms. Scale bars, 10 µm.

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  Fig. 2. Promoter function of HERV-K119 LTR. (A) Schematic representations of the HERV-K119 LTR deletions are presented as horizontal gray lines with the corresponding representative green fluorescence images of each transgenic animal. Deletion 4 results in a complete loss of GFP fluorescence. GPminer (http://gpminer.mbc.nctu.edu.tw/) prediction of regulatory motifs is depicted as short vertical lines. Blue, green, and yellow indicate TATA boxes, GC-boxes, and the strong CCAAT box, respectively. (B) Quantification of fluorescence intensities in vulva muscles of transgenic worms. ***P < 0.001. (C) Regulatory elements in the 194-bp fragment from position 675 to 868 in the HERV-K119 LTR, predicted by GPminer and JASPAR (http://jaspar.genereg.net/). The TATA box and Inr as well as different transcription factor binding sites are indicated. Human transcription factor binding sites are underlined in gray and CHE-1 is a C2H2 zinc-finger transcription factor of Caenorhabditis elegans.

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  Fig. 3. RNAi targeting Caenorhabditis elegans transcription factors. (A) Representative fluorescence images of vulva muscles in worms treated with RNAi. (B) Quantification of GFP intensities (t-test. ***P < 0.001, ****P < 0.0001). Green fluorescent protein expression is driven by HERV-K119 LTR in che-1(p696) and lin-15B(n744) mutants. (C) Images show expression of green fluorescent protein driven by HERV-K119 LTR in wild-type (C-i), che-1(p696) mutants (C-ii), and lin-15B(n744) mutants (C-iii). In lin-15B(n744) background, while most of the worms exhibit low GFP expression, very few worms expressing a high level of green fluorescent protein do not lay eggs (C-iv), and some eggs with high fluorescence do not hatch, either (C-v). (D) Green fluorescent protein levels in day-1 old transgenic worms were quantified (***P < 0.001, ****P < 0.0001, Student’s t-test).

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  Fig. 4. Phylogenetic relationships and conserved regions of CHE-1. (A) The phylogenetic tree was constructed by MEGA X (Kumar et al. 2018) with nine transcription factors. The neighbor-joining method was used with 500 bootstrap replications. Bootstrap percentages are shown next to the branches. Evolutionary distances were computed using the p-distance method and are provided in the unit number of amino acid differences per site. There were a total of 639 positions in the final dataset. (B) The amino acid alignment of C. elegans CHE-1 and human ZNF500 protein. Zinc-finger domains are shown in boxes. (C) Domain structure of LIN-15B by InterPro (Mitchell et al., 2018). LIN-15B consists of 1440 AA residues. The ribonuclease H-like (RNHL) superfamily domain (residues, 135-518) and the THAP-type zinc-finger domain (THAP-Znf, residues, 1135-1215) are indicated.