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

7. Co-immunoprecipitation To further investigate potential interactions between the progesterone receptors and the OCT4 transcription factor, rabbit anti-OCT4 was linked to resin and incubated with nuclear extract from T47D cells that have been treated sequentially with estradiol and progesterone. After washing with phosphate buffered saline, bound proteins were eluted and subjected to Western Blog analysis. As seen in Fig. 5, the rabbit monoclonal anti-OCT4 was able to pull down isoform B of the progesterone receptor (119 KD). The higher molecular weight protein (~220 KD) which was also present in the flow-through but not the serum control was presumably a non- specific signal from the nuclear extract. DISCUSSION While there has been some research on the activation of HERVs in early embryos (Grow et al. 2015, for example), our project focuses on hormonal regulation of these elements. Our major findings are: Class II HERVs are regulated by female sex hormones in T47D cells through nuclear receptors; sex hormones regulate cellular sequences through a class of solo LTRs; the effect of progesterone on HERVK expression is partly mediated by OCT4; DNA methylation has an overall effect of suppressing HERVK expression but enhances the binding of progesterone receptors on DNA. Of particular interest is the requirement of two transcription factor binding motifs in the LTR for progesterone to take effect, the palindromic progesterone-response element (GGTGCAAGATGTTCT) and the OCT4-binding element Liu and Nguyen ◀ Endogenous Retroviruses ▶ 2018 ICC 197 Fig. 4. Binding of methylated and unmethylated progesterone-response element (PRE) to progesterone receptors in nuclear extract of T47D cells. A, B, and C compare binding affinities of methylated (green) and unmethylated (red) PRE probes as revealed by competition with an unlabeled PR probe. Numbers above each column indicate the amount of unlabeled PR probe in the reaction. Unlabeled OCT4 probe served as a control for nonspecific binding. D and E show binding-kinetics of the probes using different concentrations of nuclear extract (The probes were not combined as in A, B, and C). F shows supershifiting using antibodies against OCT4 and PR (See Materials and Methods). Rabbit serum (RS) and mouse serum (MS) served as controls for nonspecific shifting effect. Figure 5. Co-immunoprecipitation of progesterone receptor and OCT4 transcription factor. Rabbit anti-OCT4 was used to pull down progesterone receptor. AntiPR was used as a positive control and rabbit serum was used as a negative control. Precipitates and flow-through were analyzed by Western Blot using rabbit antiPR as the primary antibody and HRP-conjugated goat anti-rabbit IgG. Signals were detected with enhanced chemiluminescent peroxidase substrate reagents.