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

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.

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

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

EPFL2017

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

EPFL2018

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

EPFL2018