Nup98-dependent transcriptional memory is established independently of transcription

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues has been termed transcriptional memory, which can be maintained epigenetically through cell divisions. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays, and this phenomenon has been found to depend on a nuclear pore component Nup98 in multiple species. To gain an alternative perspective on the mechanism and on the contribution of Nup98, we set out to examine single-cell population dynamics of transcriptional memory by monitoring transcriptional behavior of individual Drosophila cells upon initial and subsequent exposures to steroid hormone ecdysone. To this end, we combined single-molecule RNA FISH with mathematical modeling, and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously, begin a slow transitioning into a specialized memory state, characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation, and we were able to confirm this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98s role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.