# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 2 Results
### Epigenetic Changes in Response to Zeblararine and Dexamethasone  
The promoter regions of both _SLC34A1_ sense and _PFN3_ antisense genes contain a short CpG island with seven and six CpGs, respectively. We tested whether zeblararine and dexamethasone had an effect on the methylation status of both sense and antisense promoters (Figures 4A and S1). Quantitative pyrosequencing revealed that zeblararine induced small but significant sense promoter hypomethyl that parallels the expression changes in HKC-8 cell lines (Figure S1). A comparable reduction in sense promoter methylation was seen with HEK293 cells; in contrast, the antisense promoter stayed fully methylated (Figure 4A). Figure 3: Expression of sense and antisense transcripts in response to dexamethasone in HEK293 and HKC-8 cells. (**A**) Response of the sense transcript to increasing levels of dexamethasone in HEK293 cells (black) and HKC-8 cells (grey) measured by RT-qPCR. Human testis RNA and human kidney cell RNA were used as positive controls (right panel). (**B**) Time dependence of the dexamethasone response of sense (black) and antisense (grey) transcripts in HEK293 cells. Control RNAs from testis and kidney on the right. (**C**) Nuclear and cytoplasmic distribution of sense and antisense transcripts in HEK293 cells. Nuclear and cytoplasmic fractions were enriched followed by RNA extraction and RT-qPCR from control and dexamethasone induced cells (underscored). _XIST_ was measured to estimate nuclear enrichment, _GAPDH_ for cytoplasmic enrichment. One-way ANOVA followed by Tukey’s test for multiple comparisons, *\(p<0.05\); ***\(p<0.001\). To investigate further the epigenetic changes induced by dexamethasone, chromatin immunoprecipitation (ChIP) was performed with HEK293 cells. First, global H3 histone acetylation was shown to significantly increase by about four-fold in response to dexamethasone treatment. This effect peaked at 5 days of treatment and became reduced after 15 days but was still significantly above control (Figure 4B). To characterize histone modifications in the _SLC34A1/PFN_ promoter regions, chromatin immunoprecipitation (ChIP) was performed using antibodies against H3K27 Ac (acetylated lysine 27 of histone H3) and H3K4 Me3 (tri-methylated lysine 4 of histone H3) followed by qPCR of the _SLC34A1/PFN_ promoter regions. Both histone marks reflect active promoters. H3K27 Ac in the sense promoter region clearly increased at day 5 in response to dexamethasone whereas H3K4 Me3 remained largely unaffected. In contrast, both histone marks in the antisense promoter decreased over time and showed their lowest level after 5 days of dexamethasone (Figure 4C). To summarize, transcription at the _SLC34A1/PFN3_ locus can be activated using zebularine and dexamethasone, but the effect crucially depends on the cell lines used. The  
Figure 4: DNA methylation and histone H3 modifications of _SLC34A1/PFN3_ sense and antisense promoters in HEK293 cells. (**A**) DNA methylation levels of 7 CpGs in the sense promoter (black) and 6 CpGs in the antisense promoter (grey) after exposure to dexamethasone for 1, 5 and 15 days. Fully methylated (100%) and un-methylated fragments (0%) served as controls, methylation levels in cells exposed to dexamethasone for 1, 5 and 15 days were compared to cells without the drug. (**B**) Global histone H3 acetylation in response to the exposure of dexamethasone for 1, 5 and 15 days. (**C**) Chromatin immunoprecipitation (ChIP) using antibodies against H3K27 Ac and H3K4 Me3 followed by qPCR of sense (left panel) and antisense (right panel) promoters. Cells were exposed to dexamethasone for 1, 3 and 5 days or left without the drug as a control. Significance was established using one-way ANOVA and Tukey’s test for multiple comparisons, * \(p\) < 0.05; **_p_ < 0.01; *** \(p\) < 0.001; ns = no significance. activation is generally stronger for the _SLC34A1_ sense transcript and only just detectable for the antisense RNA.