(188df) Balancing Biophysical Tradeoffs to Drive Cellular Reprogramming

Cellular reprogramming continues to generate new cell types, increasingly expanding our perspective of cellular plasticity. However, despite improved genetic programming tools and epigenetic modulations, reprogramming remains a rare cellular event. 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 observed upregulation of transcriptional machinery and DNA repair pathways in our improved system. Specifically, 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. Topoisomerases mediate collisions between transcriptional and DNA-replicative machinery as well as resolve supercoils introduced by both processes (e.g. transcription, replication). 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 cellular reprogramming. Our observations highlight a challenge for synthetic circuits integrated into large transcriptional networks. Additionally, our work raises questions about the precise mechanisms by which hyperproliferation and hypertranscription enhance transitions in cellular state, which may refine our understanding of synthetic and pathological cellular transformations (e.g. reprogramming, cancer).