Mitigating Antagonism between Transcription and Proliferation Allows Near-Deterministic Cellular Reprogramming

Abstract

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, neardeterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.

Reconstruction of Ewing Sarcoma Developmental Context from Mass-Scale Transcriptomics Reveals Characteristics of EWSR1-FLI1 Permissibility

“…R-loops form naturally during transcription but, if unresolved, contribute to transcription-replication conflicts [39]. Without the ability to mitigate this antagonism, cells cannot maintain hyperproliferation and hypertranscription without suffering genome instability [27]. We demonstrated herein that Ewing sarcoma and PHATE_1-low cell types display the ability to resolve R-loops and replication stress.…”

“…We demonstrated herein that Ewing sarcoma and PHATE_1-low cell types display the ability to resolve R-loops and replication stress. Consequently, we propose that this mitigating capability is required to maintain the high rates of proliferation and transcription which these tissues generally display (Ewing sarcoma and pluripotent stem cells show both hypertranscription and hyperproliferation) [24][25][26][27]. However, we cannot exclude the possibility that R-loop levels, R-loop resolving factors and factors involved in responding to replication stress are independent correlates of altered proliferative state.…”

“…There exists a natural antagonism between replication and transcription [27,37]. Replication forks can stall, reverse, or collapse due to collisions with transcriptional machinery [38].…”

“…Ewing sarcoma cells display high rates of replication and transcription [24], which is unsurprising given their cancer status. However, recent reports indicate that pluripotent tissues also display these characteristics [25][26][27]. Furthermore, high rates of transcription combined with high rates of proliferation can create transcriptional stress and genome instability via the production of R-loops (genomic structures formed by the hybridization of RNA and DNA) [28].…”

Cell cycle dynamics in the reprogramming of cellular identity

“…How does this strained bioenergetic state, as represented by the Warburg state , shape the biochemical substrate pool for chromatin modulation ? On the chromatin, would the rapidly progressing replication forks collide more frequently with the transcription machineries ? What mechanisms could help to alleviate such collisions?…”