Transcription Factors and DNA Play Hide and Seek

Transcription factors (TFs) bind to specific DNA motifs to regulate the expression of target genes. To reach their binding sites, TFs diffuse in 3D and perform local motions such as 1D sliding, hopping, or intersegmental transfer. TF-DNA interactions depend on multiple parameters, such as the chromatin environment, TF partitioning into distinct subcellular regions, and cooperativity with other DNA-binding proteins. In this review, how current understanding of the search process has initially been shaped by prokaryotic studies is discussed, as well as what is known about the parameters regulating TF search efficiency in the context of the complex eukaryotic chromatin landscape.

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

Coordinated Effects of Sequence Variation on DNA Binding, Chromatin Structure, and Transcription

Julien Bryois, Bart Deplancke, David Hacker, Nouria Hernandez, Sunil Kumar Raghav, Sarah Thurnheer, Gilles Udin, Sebastian Martin Waszak, Alisa Yurovsky

DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.

American Association for the Advancement of Science2013

Mitotic chromosome binding predicts transcription factor properties in interphase

Mahé Cécile Raccaud

EPFL2019

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