Eukaryotic transcription factor binding kinetics

Transcription represents a key regulatory step in gene expression and transcription factors (TFs) are the key molecular players of this process. TFs inhibit or promote the assembly of the transcriptional machinery by binding DNA and several co-factors. Whereas biochemical studies suggested stable TF-DNA interactions (timescales of hours), fluorescence microscopy showed that TFs bind and dissociate from DNA within few seconds. However, the biological implications of such a dynamic behaviour are still elusive. TF-DNA binding kinetics have been mainly measured in interphase cells, i.e. when active transcription occurs. Though recent findings suggest that even when transcription is silenced in mitosis, some TFs bind to chromatin, a phenomenon known as âmitotic bookmarkingâ. This mechanism was proposed to regulate phenotypic maintenance by ensuring faithful gene reactivation at the mitosis-G1 transition. At present, though, evidence on whether and how binding kinetics determine TFsâ mitotic chromosome binding is missing. In this work, we aimed to determine the role of TFs-DNA binding kinetics in two functional aspects: (i) transcriptional activation of genes involved in the inflammatory response; (ii) differential mitotic chromatin binding of TFs regulating reprogramming and differentiation. We investigated the first aspect by measuring the binding kinetics and transcriptional activity of p65-NFï«B and mutants to its DNA-binding domain (DBD) and transactivation domains (TADs). By performing single-molecule tracking (SMT) of fluorescent p65 molecules and extrapolating p65 transcriptional activation potentials from RNAseq, we demonstrated that p65 binding time (t_b) generally correlates to its capability to induce transcriptional activation. Unexpectedly, deletion of p65 transactivation domains (TADs) did not significantly reduce p65 t_b (~3-6 s) if compared to wt-p65 (t_b~4 s), although it strongly inhibited p65-mediated transcriptional activation. We also showed ~1000-fold reduction of DNA-binding affinity (Y36A/E39D mutant) retained ~3% of long-lasting binding events (t_b~12 ð ) linked to no-activity. This prompted us to speculatively suggest that p65 may bind DNA indirectly. In a second part, we completed the kinetic characterization of Sox2 and Oct4, two TFs of the differentiation/reprogramming network. Preliminary findings showed that Sox2 and Oct4 display different mitotic chromosome binding and comparable dissociation rates from mitotic chromatin. Thus, we tested whether their distinct retention on mitotic chromosomes could arise from different association rates. To test this hypothesis, we performed SMT of Sox2- or Oct4-Halo in ES living cells to compute their bound fraction during different phases of their cell cycle. Sox2 and Oct4 were equally retained on interphase cells and did not show significant differences even during mitosis, suggesting that neither dissociation nor association rates at specific binding sites determine the differential retentions of the two TFs. Our findings indicate that (i) p65 binding kinetics predict functional outcomes as long as protein-protein interactions are preserved; (ii) such interactions do not stabilize p65-DNA binding but critically define binding events as transcriptionally productive; (iii) differential retention of Sox2 and Oct4 on mitotic chromatin is not due to differences in their binding kinetics at specific sites, suggesting a contribution from TFs-DNA non-specific interactions.

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

Mitotic chromosome binding predicts transcription factor properties in interphase

Mahé Cécile Raccaud

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape to regulate transcription is unclear. TFs also differ in their ability to associate with mitotic chromosomes, which has been shown to be mediated largely by non-specific binding. The TFs identified so far for their ability to bind mitotic chromatin are known cell fate regulators and often have been shown to have pioneering activity (i.e. they are able to bind and open closed chromatin regions). However, the number of TFs tested for mitotic chromosome binding (MCB) and the knowledge about properties allowing TFs to bind to mitotic chromatin are limited. Thus, we performed a large-scale quantification MCB by TFs. Out of 502 quantified TFs, we observed that 22% of TFs were enriched on mitotic chromosomes, 24% were depleted and 54% were neither enriched nor depleted, and these behaviors were largely cell-type non-specific. As TF-DNA interactions are mediated by their DNA-binding domains, we investigated their influence on MCB. However, despite the fact that TFs bearing certain types of DNA-binding domains were more likely to be enriched or depleted from mitotic chromosomes, it was not sufficient to explain differential MCB. Amino acid sequence-based analysis using machine learning showed that electrostatic properties impact the propensity of TFs to bind mitotic chromosomes. This confirms that co-localization of TFs with mitotic chromosome is affected by non-specific electrostatic protein-DNA interactions. Therefore, we next investigated whether the MCB reflected properties of TFs in interphase. We observed a clear correlation of MCB with DNA co-localization in interphase fibroblasts and with mobility in both interphase and mitosis. As mobility is influenced by both TF diffusion and binding to DNA, we excluded that differences in diffusion, caused by TF sizes, could explain the variability in mobility rates, and tested whether TFs differed in their association with specific DNA sites. Single molecule imaging of DNA binding in live cells showed that differences in MCB were correlated with differences in relative TF on-rates, but not with TFs residence times. To test whether this affected target site occupancy, genome-wide mapping of TF binding was performed, showing that TFs associating to mitotic chromosomes also have a higher interphase genome occupancy and greater ability to find their specific sites in the genome. Lastly, we investigated the correlation between the abilities to bind mitotic chromatin and to modify chromatin accessibility and observed that the broader impact of TFs with a high MCB on chromatin accessibility can be explained by their high number of bound target sites, and not by a higher potency to alter chromatin accessibility. Altogether, our data suggests that TFs differ broadly in their non-specific DNA binding properties, which regulate their search efficiency and thereby their occupancy of specific sites in the genome. We suggest that measuring mitotic chromosome association of TFs by fluorescence microscopy can be used as a metric of non-specific DNA binding.

EPFL2019

Chromatin organization, transcription factor dynamics and gene expression in higher eukaryotes with superresolution imaging and single molecule tracking

Aleksandr Benke

Gene expression is dynamic and heterogeneous across all cell types. It serves several fundamental functions such as adaptation for changing environment and developing tissue specific phenotype and functionality during development and in adult organism. Control of gene expression mostly occurs at the transcription stage. Two main factors that regulate transcription in high eukaryotes are chromatin organization and transcription factor (TF) protein dynamics. Until recently they were mostly studied by methods that are performed in vitro or/and require ensemble averaging. We have been contributing to a development of new single molecule imaging methods that allow quantifying chromatin organization and TF dynamics in vivo and at single cell/single molecule level. Here we present our methodological developments and apply them to study how chromatin organization and TF dynamics regulate transcription. On the technical side of the study we, first, developed a method of imaging DNA with enhanced resolution in living cells. Super-resolution (SR) point localization based microscopy allows visualizing sub-diffraction organization of cellular structures and their dynamics in vivo. However, until recently almost exclusively proteins were imaged in SR microscopy. We found photoswitching conditions to image with stochastic optical reconstruction microscopy (STORM) DNA directly using the site-specific DNA –binding dye Picogreen (Chapter 2). We achieved a resolution of 50-70 nm which is ~5 fold better than conventional microscopy. Due to the excellent dye preservation we were able to do time lapse SR imaging. This study was a first demonstration of using a site-specific dye for STORM SR imaging in living cells. Photoswitching principle used in SR microscopy can be applied to single molecule (SM) tracking. It provides up to 1000x increase in density of trajectories compared to classic SM tracking. Initially, photoactivatable (PA) fusion proteins were used for high density SR based tracking. However, PA proteins are relatively dim and there is a limited choice of them. We demonstrated a new approach to SR based high density tracking by using organic dyes’ photoswitching to track proteins (Chapter 4). We showed that different dyes can be photoswitched in similar live-cell compatible media on membrane and in different organelles. Together with orthogonal protein labelling schemes this allows us to perform multicolour high density tracking across different compartments. This approach gives flexibility of labelling and increased track length compared to PA-FP SR based tracking. In a separate study, using tracking we developed algorithm which can account for long-living molecules in live cell SR imaging and allows us to correct clustering artefacts (Chapter 5). [...]

EPFL2015

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

Torgil Elias Friman

EPFL2019

Chromatin organization, transcription factor dynamics and gene expression in higher eukaryotes with superresolution imaging and single molecule tracking

Aleksandr Benke

EPFL2015

Mitotic chromosome binding predicts transcription factor properties in interphase

Mahé Cécile Raccaud

EPFL2019