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

labeled probes and incubated at room temperature for 7-8 minutes. For supershifting, 1 µL of undiluted mouse monoclonal antiPR (Fischer, MA1412) or rabbit monoclonal anti-OCT4 (Boster, M00174) was added to the reaction and incubated for 20 minutes at room temperature or 10 minutes at room temperature followed by 1 hour on ice. Normal mouse plasma and normal rabbit serum served as controls for mouse antiPR and rabbit anti-OCT4. Concentration of dithiothreitol was reduced to 2 mM for antiPR and mouse plasma, and 1.5 mM for anti-OCT4 and rabbit serum. Polyacrylamide gel electrophoresis was carried out using non-denaturing Mini- PROTEAN TBE Gels (BIO-RAD). Oligonucleotide bands were visualized using an Odyssey CLx scanner. 10. Co-immunoprecipitation and Western blotting Pierce Co-Immunoprecipitation Kit (Thermo Scientific) was used. Ten micrograms of rabbit anti-OCT4 or rabbit antiPR monoclonal antibodies (Boster) were linked onto AminoLink Plus Coupling Resin. Rabbit serum was used as negative control. Linked antibodies were allowed to react with nuclear extract from T47D cells stimulated sequentially with estradiol and progesterone. After washing resin with phosphate-buffered saline, proteins were eluted, separated with an Any kD™ Mini-PROTEAN ® TGX™ Precast Protein Gel, and transferred onto a nitrocellulose membrane using the Mini Trans-Blot ® Cell (Bio-Rad). Progesterone receptor proteins were detected using rabbit antiPR and the Enhanced Chemiluminescent Reagent Kit (Boster). 11. Statistical analysis All quantitative data were analyzed with the Student’s t-test. Statistical significance is defined at P < 0.05. RESULTS 1. Female sex hormones enhance HERVK expression in T47D cells through nuclear receptors Although the T47D breast cancer cell line has been known to express human endogenous retroviruses in response to female sex hormones, the underlying mechanisms of hormonal induction have not been studied (Golan et al. 2008; Patience et al. 1996; Ono et al. 1987) According to a protocol established by Ono et al., we treated T47D cells with estradiol for 24 hours followed by a combination of estradiol and progesterone treatment for 13-15 hours. Using quantitative reverse-transcription PCR, we analyzed the expression of various HERV elements. Consistent with previous findings, we were able to demonstrate hormonal activation of HERVK genes in the cell line (Fig. 1A and 1B, P < 0.05). The effect of progesterone is dose-dependent (Fig. 1C). Either an estrogen antagonist, fulvestrant, or a progesterone antagonist, mifespristone, can block the enhancing effect of the hormones. Because these drugs work on nuclear receptors, it is evident that estradiol and progesterone activate HERVK through nuclear receptors. Testosterone or cortisol had no effect on the expression of HERVK env (data not shown). Patience et al. (1996) reported that only HERVK10-like sequences were discovered in retroviral particles produced by hormone- stimulated T47D cells. Wang-Johanning et al. (2001) failed to detect expression of ERV3 or HERV-E4-1 in breast tumor tissues. However, well-known HERV elements involved in human reproduction are mostly class I HERVs, not HERVKs. To study the expression of representative class I HERVs in response to female sex hormones, we quantified the transcripts of syncytin-1 and syncytin-2, which are env genes of ERV and ERVFRD, respectively, and the pleiotrophin gene, which is a cellular gene whose expression in trophoblasts is driven by a truncated ERV (Ball et al. 2009; Schulte, et al. 1996). All three genes are known to be involved in placental development, and therefore may be influenced by female sex hormones. Using quantitative PCR, we found appreciable expression of all three genes in T47D cells. Consistent with the findings of Patience et al., expression of none of these genes were altered by hormonal treatments (data not shown). 2. Female sex hormones regulate host gene expression through solo LTRs Gogvadze et al. (2003) reported that two solo LTRs, both belonging to the LTR5HS subgroup, drove expression of downstream host DNA sequences. Both reside in introns on the noncoding strand, initiating transcription of antisense RNA against the host gene. One of them is located between exons 5 and 6 of a gene named Solute Carrier Family 4 Member 8 (SLC4A8), and the other is located between exons 23 and 24 of a gene called Intraflagellar Transport 172 (IFT172). Quantification of the LTR-driven transcripts in the brain showed a difference between human male and female samples, as well as a difference between the human samples and a chimpanzee sample (Gogvadze et al. 2003). The T47D cells provide a model to investigate the effect of female sex hormones on gene expression driven by solo LTRs. Using primers designed by Gogvadze et al., we indeed found a stimulating effect of hormonal treatment on LTR-driven expression of downstream host sequences, specifically antisense RNA against exons of SLC4A8 and IFT172 (Fig. 1D, P < 0.01 for SLC4A8). Mifepristone, a progesterone receptor antagonist, blocked the effect of female sex hormones completely. We found in T47D cells that the LTR5HS in SLC4A8 is a stronger promoter/enhancer than the LTR5HS in IFT172 (Fig. 1D). This is interesting because Gogvadze et al. (2009) reported higher expression of the IFT172 antisense RNA in normal testes and seminoma (germ cell tumor of testis) specimens. It indicates that promoter/enhancer activities of different versions of LTR5HS may be tissue-specific and/or gender-specific. 3. LTR5HS drives expression of reporter genes and responds to sex hormones in T47D cells In order to investigate the molecular mechanisms of the action of steroid hormones on the HERV LTR, we cloned LTR5HS and LTR13A upstream of a luciferase reporter gene. The LTR5HS was amplified from the SLC4A8 intron at 12q13.13, as described previously, while LTR13A was amplified from the first intron of a gene called Inositol 1,4,5-Trisphosphate Receptor Type 3 (IPTR3) at 6p21.31. Upon transfection into T47D cells, we found that estradiol and progesterone upregulated the promoter/enhanced activity of LTR5HS but not that of LTR13A (Fig. 2A). In the absence of progesterone, the effect of both LTRs on the expression of the reporter gene was comparable to that of the vector alone. Using LTR5HS from HERVK-con (Lee and Bieniasz 2007), Grow et al. (2015) found that the promoter/enhancer activity of Liu and Nguyen ◀ Endogenous Retroviruses ▶ 2018 ICC 193