# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 2 Results
### Drug-Induced Expression of SLC34A1/PFN3 Sense-Antisense Transcripts  
Natural sense/antisense transcripts are often co-expressed in the same RNA preparations [30]. To assess potential co-expression _SLC34A1/PFN3_ transcripts, we used the established human kidney cell lines HEK293 and HKC-8, and drugs that modify the epigenetic imprint to identify conditions that lead to detectable transcripts from the _SLC34A1/PFN3_ locus. Cells were exposed to zebularine (DNA methyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor) and dexamethasone (broad effect on both DNA methylation and histone modifications) followed by expression analysis of _SLC34A1/PFN3_ sense and antisense transcripts by RT-qPCR. The primers used to amplify fragments of _PFN3_ only detected the splice form that overlaps the _SLC34A1_ gene. In HKC-8 cells, the _SLC34A1_ sense transcript was significantly enhanced in response to zebularine as compared to non-treated cells; dose-response experiments established 50 uM zebularine as the most effective dose (Figure 2A). The increase in HKC-8 cells was time-dependent, and it plateaued after 48 h incubation (Figure 2B). A comparable response was measured in HEK293 cells, but the increase in mRNA was not significant (Figure 2C). The _PFN3_ antisense transcript was only minimally expressed in HK-8 cells, and detectable after 24 h incubation with the drug (Figure 2B). Trichostatin A, on the other hand, did not provoke any detectable output from the _SLC34A1/PFN3_ locus, despite global acetylation levels rising significantly after  
Figure 1: Architecture and expression of the _SLC34A1/PFN3_ (sense/antisense) locus. **(A)** Modified screenshot of the human _SLC34A1/PFN3_ locus in the UCSC genome browser. The _SLC34A1_ sense gene and the _PFN3_ gene in antisense are at the top, published human [7] and murine [23] antisense transcripts are below. Conservation in 100 vertebrates (light blue) and CpG islands (green). **(B)** Expression of _SLC34A1_ and _PFN3_ mRNA and protein in various organs adapted from the human protein atlas ([[https://www.proteinatlas.org/](https://www.proteinatlas.org/)]([https://www.proteinatlas.org/](https://www.proteinatlas.org/)), accessed on 10 December 2021). **(C)** Expression of sense (_SLC34A1_, black bars) and antisense transcripts (_PFN3_, grey bars) was assessed by RT-qPCR in human testis RNA and RNA extracted from primary kidney cells. The log2 fold change refers to uninduced HKC-8 cells. drug treatment (Figure S2). Moreover, trichostatin A and zebularine showed no signs of synergistic stimulation (not shown). Dexamethasone (100 nM) provoked the most prominent transcriptional stimulation of the _SLC34A1/PFN3_ locus in HEK293 cells; a comparably small response was observed in HKC-8 cells under identical conditions (Figure 3A). The drug increased the expression of the sense transcript in a time-dependent manner up to about 15-fold after five days; in parallel, antisense transcript levels were also enhanced by approximately 3-fold (Figure 3B). Separation of nuclear and cytoplasmic cell fractions revealed enrichment of the sense transcript in the cytoplasm whereas the small amount of antisense transcript was confined to the nucleus (Figure 2C). The sense transcript followed the enrichment pattern of the cytoplasmic control _GAPDH_ whereas the antisense transcript paralleled _XIST_ nuclear enrichment. Figure 2: Expression of the _SLC34A1/PFN3_ (sense/ antisense) locus in in response to zebularine. The expression of sense (_SLC34A1_) and antisense transcripts (_PFN3_) was assessed by RT-qPCR; human tests RNA and RNA extracted from primary kidney cells were used as controls (right panels of (**A–C**)). (**A**) Response of the sense transcript to zebularine in HKC-8 cells and (**B**) HEK293 cells. (**C**) The most effective dose was used (50 nM) to establish the time course of expression for both the sense (black) and the antisense (grey, only detectable at 24 h) transcripts.