(490e) Quantitative Analysis on LIF Signaling- and Epigenetics-Mediated Transcriptional Gene Regulation in Mouse Embryonic Stem Cells

It is shown that both epigenetic states and cell signaling pathways have significant effects on cell reprogramming efficiency. However, previous techniques relied on fixed cells across cell population, lacking temporal resolution and failing to account for cell-to-cell variability. The kinetics of how the activation of the signaling pathway or epigenetic modifications affect downstream gene expression dynamics remain unclear. We utilized CRISPR-mediated live imaging to investigate how LIF signaling pathway and epigenetic modification affect the downstream gene expression dynamics in mouse embryonic stem cells (mESCs). To visualize endogenous transcriptional activity of key embryonic genes Sox2 and Klf4, we inserted 24 copies of the MS2 or PP7 to the endogenous Sox2 and Klf4 gene loci using CRISPR/Cas9 system. Each MS2/PP7 repeat forms a stem-loop structure, which recruits two copies of the corresponding coat protein fused with fluorescent proteins. This allows visualization of the nascent transcript of target genes in a high spatiotemporal resolution.

We first modulated the LIF pathway activity by adding 10µM JAK inhibitor I or by removing LIF from the media. Cells were imaged every 24 hours after each perturbation to characterize how fast the genes could respond to the cell signaling changes. Our results showed that the fraction of cells showing the active Sox2 signal decreased 48hr after JAKi addition and 72hr after LIF removal. This time discrepancy is likely attributed to the slow degradation of LIF and high binding efficiency of JAK inhibitor. Moreover, it was found that the cells that lost Sox2 signal exhibited more signs of cell differentiation with two heterochromatin structures near the nucleolar. We also found that total mRNA output of Sox2 was significantly reduced 48hr after LIF removal. A two-state model of transcriptional bursting was implemented to characterize the transcriptional kinetic parameters in single cells. By converting signal intensity trajectories of each cell to a binary promoter ON and OFF state, we found that the reduction of total mRNA output after LIF removal was predominantly caused by a decrease in bursting frequency.

To further analyze pluripotent gene expression dynamics in response to epigenetic changes, we developed a reversible and inducible CRISPR/dCas9-based tool to modulate epigenetic states on a specific locus in living cells. Histone modifiers such as HP1α were recruited to induce histone methylation on the target locus (Sox2 and Klf4) in the presence of the ABA inducer. We measured the changes in histone methylation level over time through ChIP-PCR using histone modification antibody. With live imaging, we were able to characterize the time scale of epigenetic changes and the kinetics of Sox2/Klf4 gene expression in single cells. Our work provides a quantitative and single-cell analysis on the impact of signaling pathway modulation in transcription and in subsequent cell reprogramming.