# Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (_SLC34A1_/Pfn3)
## 3 Discussion  
We report a cell culture system that allows manipulating the expression of the _SLC34A1/PFN3_ sense-antisense transcript pair using dexamethasone and CRISPR gene editing. In essence, we found that sense and antisense transcripts are positively co-regulated, and mRNA levels are paralleled by epigenetic marks, i.e., DNA methylation and histone modifications in sense but not antisense direction. The observed effects are cell line specific, as generally observed with other regulatory lncRNAs. The most striking feature of natural antisense transcripts is their obvious regulatory potential related to complementary sense transcripts. Hence, a sense-antisense arrangement of gene pairs was hypothesized to provide an evolutionary advantage, possibly through one of a few specific, conserved molecular mechanism(s) [12; 32]. In fact, several early observations suggested that such antisense-mediated regulatory mechanisms indeed existed. For example, sense and antisense transcript pairs were found in the same cDNA libraries [6]. Convergent genes show hallmarks of evolutionary conservation [9] and potentially common structural features [26], and natural antisense transcripts are depleted in the mammalian X chromosome [5; 6]. However, testable predictions deduced from hypothesized regulatory mechanisms, for example specific functional or structural properties of bi-directionally transcribed genes or endo-siRNA formation, gathered only limited experimental support [33; 34]. Output and outcome of sense-antisense transcription and transcript interactions depend largely on the model system where the co-expression is studied. Both stimulatory and inhibitory effects of antisense transcripts on sense transcript expression are now widely documented, as is independent regulation of the two complementary genes [8; 35]. We report a cell culture model with inducible sense and antisense gene expression. With both dexamethasone stimulation and CRISPR-mediated transcriptional activation, convergent expression of sense and antisense RNAs was observed. The observed effects only became detectable after extended incubation time (5 days), though other systems have been reported where epigenetic changes and transcription of the affected locus were detected in a much shorter time frame of minutes [36]. The effect of dexamethasone was shown to be dependent on transcription rather than RNA-mediated, since trans-activation from transfected DNA constructs was not observed. Moreover, comparable  
Figure 7: Functional expression of _SLC34A1_ in _Xenopus_ oocytes and HEK293 cells. (**A**) Wild type HEK293 cells were grown with and without dexamethasone for 5 days, followed by uptake measurement using radioactive Pi. Values were scaled to an average control value of 100%, \(t\)-test revealed significance, \(p\) < 0.01. (**B**) RNA was extracted from HEK293 cells, untreated (WT) and after 5 days exposure to dexamethasone (WT + dex). The same procedure was carried out with HEK293 cells that had biallelic insertion of the antisense cassette (AS biallelic KO + dex). Total RNA was injected into _Xenopus_ oocytes, and the uptake of radioactive Pi was measured after 3 days. One-way ANOVA followed by Tukey’s test for multiple comparisons, \(p\) < 0.01.  
effects elicited by different stimulatory interventions (dexamethasone and CRISPR) suggest that transcription itself rather than an interaction of the promoters provides the stimulatory drive. The activation was reflected by reduced promoter methylation and activating histone marks (H3K27 Ac) at the sense promoter, less so at the antisense promoter. Accordingly, the stimulatory effect of dexamethasone and sense transcription on antisense expression was weak. Coordinated expression of clustered genes is a well-documented phenomenon, originally described as 'transcriptional ripples' [37; 38]. The transcriptional co-regulation of locally connected genes often does not feed through to the protein level, i.e., transcriptome changes are not reflected at the proteome level [39].