Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.

The role of OCT4 and SOX2 in regulating chromatin accessibility and cell fate across the cell cycle in embryonic stem cells

Torgil Elias Friman

Precise spatiotemporal regulation of gene expression is essential for development and homeostasis of complex organisms. This is achieved in large part by sequence-specific transcription factors (TF) that bind to genomic regulatory elements to activate or repress transcription. DNA in eukaryotic nuclei is wrapped around histones into nucleosomes, which are inaccessible to most proteins. However, certain TFs can access their binding sites in the presence of histones and promote nucleosome eviction to increase chromatin accessibility, allowing for binding of other protein. These TFs, called pioneer factors, include OCT4 and SOX2, which are master regulators of embryonic stem (ES) cells. Dividing cells, including stem cells, must achieve proper gene regulation despite the progression of the cell cycle, which imposes several constraints. During S phase, the replication fork passes through the genome to make a copy of each chromosome, which leads to a loss of chromatin accessibility. Furthermore, the substantial chromatin compaction that occurs during mitosis to prepare for chromosome segregation is associated with eviction of most DNA-binding proteins and reduced accessibility at distal cis-regulatory elements including enhancers. How pioneer factors operate in the context of cell cycle progression is unclear. In this thesis, I will present work studying different features of OCT4 and SOX2, including how they interplay to regulate chromatin accessibility in ES cells, the impact of small endogenous fluctuations of their protein levels on cell fate and chromatin accessibility, and how their roles in regulating cell fate and chromatin accessibility is related to different stages of the cell cycle. Using live-cell imaging of fluorescently labeled proteins, we discovered that OCT4 and SOX2 were associated to mitotic chromatin. Using cell-cycle specific degradation strategies, we could show that the presence of SOX2 and OCT4 at the mitosis-G1 (M-G1) transition contributes to maintain the pluripotency of ES cells. We further showed that ES cells with high levels of OCT4 in G1, but not in S phase, are more prone to differentiate towards neuroectoderm and mesendoderm cell fates. Cells with high levels of OCT4 displayed increased chromatin accessibility at enhancers of differentiation genes, suggesting accessibility fluctuates with OCT4 levels and can prime cells for differentiation. To explore potential cell cycle-dependent regulation of chromatin accessibility, we degraded OCT4 at the M-G1 transition, which led to a reduction in accessibility at many OCT4-bound regions. When OCT4 returned later in the cell cycle, chromatin accessibility was recovered, although at many loci this recovery was incomplete, suggesting that OCT4 is required at the M-G1 transition to maintain these regions open. We then rapidly degraded OCT4 at other phases of the cell cycle. Surprisingly, loss of chromatin accessibility was highly similar at all cell cycle phases, showing that OCT4 is constantly required to maintain regulatory elements accessible. To explore the dynamic regulation of accessibility by OCT4, we performed time-course measurements of accessibility upon its degradation. This revealed that loss of accessibility was temporally correlated to the degradation of OCT4. These results suggest that chromatin accessibility is highly dynamic and continuously dependent on the presence of OCT4 across the cell cycle in ES cells.

EPFL2019

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018

A role for mitotic bookmarking of SOX2 in pluripotency and differentiation

Aleksandr Benke, Andrea Callegari, Cédric Deluz, Torgil Elias Friman, Marion Leleu, Suliana Manley, Mahé Cécile Raccaud, Daniel Strebinger, David Michael Suter

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [Chill combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.