Lende-Dorn, B. A. | bioRxiv Science

bioRxiv2024

Chemogenetic tuning reveals optimal MAPK signaling for cell-fate programming

Cell states evolve through the combined activity of signaling pathways and gene regulatory networks. While transcription factors can direct cell fate, these factors rely on a cell state that is receptive to transitions in cell identity. How signaling levels contribute to the emergence of receptive cell states remains poorly defined in primary cells. Using a well-defined model of direct conversion, we examined how levels of the MAPK-activating oncogene HRASG12V influence direct conversion of primary fibroblasts to induced motor neurons. We demonstrate that an optimal Goldilocks level of MAPK signaling efficiently drives cell-fate programming. Rates of direct conversion respond biphasically to increasing HRASG12V levels. While intermediate HRASG12V levels increase the rate of conversion, high levels of HRASG12V induce senescence. Through chemogenetic tuning, we set optimal MAPK activity for high rates of conversion in the absence of HRAS mutants. As MAPK pathways influence cell-fate transitions in development and disease, our results highlight the need to tune therapeutic interventions within a non-monotonic landscape that is shaped by genetics and levels of gene expression. HighlightsO_LIMAPK signaling drives proliferation and conversion of fibroblasts to motor neurons C_LIO_LICell-fate programming responds biphasically to HRASG12V expression C_LIO_LIHigh HRASG12V expression induces senescence, which reduces conversion C_LIO_LIChemogenetic tuning of MAPK activity increases conversion rates C_LIO_LIA small-molecule MAPK inducer drives high rates of conversion in the absence of HRASG12V C_LI

systems biology↗

bioRxiv2023

Proliferation history and transcription factor levels drive direct conversion

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies. One Sentence SummaryUsing a systems and synthetic biology approach to study the molecular determinants of reprogramming, we find that proliferation history and transcription factor levels drive cell fate in direct conversion to motor neurons. HighlightsO_LIminimal high-efficiency cocktail allows systematic interrogation of the reprogramming process C_LIO_LIhistory provides a principal axis to distinguish transcription factors influence C_LIO_LIof the transcription factor cocktail impacts reprogramming efficiency and dynamics C_LIO_LIof individual transcription factors differentially influence the rate of reprogramming C_LIO_LIearly hyperproliferation increases direct conversion of adult human fibroblasts C_LIO_LIptimal cocktail allows neurotrophic factor-free reprogramming and in vivo grafting C_LI

cell biology↗