Slick Software, Slow Hardware: Balancing Biophysical Tradeoffs to Drive Cellular Reprogramming

One challenge in synthetic biology is to integrate synthetic circuitry into larger transcriptional networks to mediate predictable cellular behaviors. While significant efforts have been devoted to the design of enhanced synthetic circuitry, less is understood regarding how cellular hardware (e.g. transcriptional processing machinery) may impose fundamental performance limitations on integrated circuits. Within the mammalian context, cellular reprogramming continues to generate new cell types, increasingly expanding our perspective of cellular plasticity. However, despite improved genetic tools and epigenetic modulations, reprogramming remains a rare cellular event. We find that cellular reprogramming is limited to a small population of cells equipped to process the massive transcriptional realignment induced by transcription factor overexpression. Previous work in iPSC reprogramming and our work in post-mitotic neurons identified fast-cycling cells as a privileged population within the converting culture. However, reprogramming of this privileged population remains non-deterministic. Given that successful conversion requires cells to undergo a massive transcriptional realignment, we took a systems-level approach to examine the connection between transcription and proliferation during conversion. We find that transcription factor-induced hypertranscription antagonizes cellular proliferation and hyperproliferating cells have lower transcription rates. Given this inherent antagonism, hypertranscribing, hyperproliferating cells (HHCs) represent a rare cellular state. We identified a combination of chemical and genetic methods capable of expanding the population of HHCs and inducing a 100-fold increase in conversion rate to induced motor neurons (iMNs) from both mouse and human primary fibroblasts. By profiling cells early in conversion, we identified topoisomerase expression (e.g. Top1, Top2A) as a key parameter modulating the cell’s ability to sustain the population of HHCs and induce reprogramming. Our data suggest that limits in reprogramming arise from tradeoffs between the inherent antagonism between transcription and replication rates. Increasing expression of topoisomerases expands the cell’s ability to mediate conflict between these two processes, enabling robust cellular reprogramming.