The Proceedings of the Eighth International Conference on Creationism (2018)

LTR5HS was dependent on an OCT4 motif which is located about 500 bp downstream of the conserved androgen-response element/ progesterone response element/glucocorticoid response element (Manghera and Douville 2013). With the plasmids constructed by Grow et al., we demonstrated that responsiveness to sex hormones is completely abolished if the OCT4 motif is deleted (Fig. 2B). We also discovered a surprising effect of DNA methylation on the responsiveness of LTR5HS to sex hormones. If the plasmids were grown in the Stellar Competent Cells (Clontech), we failed to see the effect of sex hormones on LTR5HS. The above mentioned effects were only visible when the plasmids were grown in a non- methylating strain of Escherichia coli (Stellar Competent Cells, dam - /dcm - ). Indeed, we observed a repressive effect of hormones when the OCT4 motif was mutated (Fig. 2C). Even though bacterial methylation of DNA is very different from that of eukaryotic cells, this prompted us to look into the interplay between DNA methylation and the action of hormones. 4. Knocking down OCT4 reduces expression of HERVK elements To further investigate the role of OCT4 in activation of HERVK elements, we used specific siRNA to knockdown OCT4 expression in T47D cells. OCT4-specific siRNA reduced the expression of HERVK10 env for about 70%, although transfection with random duplex RNA also significantly reduced expression of env (Fig. 3A). Similarly, expression driven by the solo LTR in the SLC4A8 gene was inhibited by OCT4 knockdown, although the difference between specific siRNA and random duplex RNA did not reach statistical significance (data not shown). Liu and Nguyen ◀ Endogenous Retroviruses ▶ 2018 ICC 194 Figure 1. Effect of estradiol and progesterone on HERVK and adjacent genes in T47D cells. A. Effect of hormones and antagonists on the expression of HERVK10 env gene. Cells were treated with estradiol (10 nM) and progesterone (1000 nM) in the absence or presence of antagonists. Total RNA was reverse transcribed and quantitative RT-PCR was performed using primers targeting the env gene of HERVK10 elements. Transcript quantity was normalized against that of the YWHAZ housekeeping gene using the Pbfaffl method. * denotes statistical significance compared with untreated control. ** denotes significant difference compared with sample without antagonists. B. Similar to A except using degenerate primers targeting the pol gene of all HERVK elements. C. Similar to A except using a gradient of progesterone concentrations. D. Similar to A except using primers targeting transcripts of host DNA downstream of solo LTRs, and the progesterone concentration was 100 nM. Bottom of D show locations of LTRs and PCR primers (adapted from Gogvadze et al. 2003). Quantitative PCR was performed in triplicates or duplicates.