Numerous studies showed that occurrence of sense-antisense gene pairs is quite common in human and other genomes. Such pairs are often formed by two protein-coding genes however, with the discovery of antisense lncRNAs, the interest in sense-antisense overlaps between protein-coding genes significantly decreased. Nevertheless, considering recent advances in sequencing technologies we reinvestigated the phenomenon of this very specific case of antisense transcription. In our studies we utilized TSS-seq data from 73 human and 13 mouse libraries and performed multilevel analysis to examine overlap conservation across species, various tissues, experimental conditions and individuals. We identified 592 human and 113 mouse gene pairs that overlap at their 5‘ end in at least one library. None of them overlapped in all libraries and majority utilized overlapping TSSs in selected tissues/cell lines only. Our results show that 5’ overlap between two genes is highly instable. This instability is not just a simple consequence of tissue specific factors. The switch between two states, non-overlapping and overlapping, very often reflects the response of the cell to changing environmental conditions. We identified, for example, 15 gene pairs that shifted from non-overlapping to overlapping state in reaction to a transfection. Moreover, analysis of 26 adenocarcinoma cell lines showed that overlap between two genes might not be conserved even in cell lines of the same type and cultured under the same conditions. In addition, we showed that overlap between genes does not have a negative effect on their expression. On the contrary, genes have higher expression level in tissues/cell lines in which they overlap while compared with the expression in cell lines where both genes are expressed but do not overlap. However, as our results show, this elevated expression is not directly related to the fact that genes overlap but is resulting from the acquisition of additional promoters.