Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-B (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an enhanceosome, nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription. Author summary To control the rate of transcription of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TF), that bind promoter sequences to mark them for the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to investigate these hypotheses using the transcriptional activator NF-B (p65) as well as mutants affecting one of its functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relation depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, transcriptional output relies upon functional protein-protein interaction subunit.

Quantitative single cell dynamics of signaling and transcriptional response in mammalian cells

Onur Tidin

Cells live in ever-changing environments, thereby facing a variety of dynamic environmental signals. Environmental stimuli elicit intracellular responses through signaling pathways, which converge on transcriptional activation or repression of target genes. Despite intensive research, dissecting the complex interactions between pathway components that modulate mRNA and protein production still remains a difficult task. To this end, single-cell approaches provide unique insights into intracellular processes and cell responses to environmental stimuli, otherwise inaccessible with traditional bulk studies. Single-cell measurements have revealed that isogenic cells sharing the same environment display substantial heterogeneity in both transcript and protein level. As the initial step in gene expression, transcription is an important source of such variability in eukaryotic cells due to low number of molecules involved, transcription factor dynamics and the discrete nature of biochemical reactions. Notably, production of mRNA during transcription occurs in short periods of activity, termed as transcriptional bursts, followed by longer periods of inactivity. Despite widespread observations of transcriptional dynamics in mammals, upstream molecular mechanisms shaping the eukaryotic transcription remained elusive. In this study, we quantitatively linked upstream factors and transcriptional kinetics by developing an experimental system to temporally monitor, simultaneously, inputs (transcription factors) and outputs (target gene expression) in mammalian cells, in response to stimulus. We established two Tet-On inducible stable cell lines each expressing a fusion protein of a luminescence (Nanoluciferase) reporter to either SMAD4 or SMAD2, the two main transcrip- tion factors downstream of the TGF-Î² pathway. These cell lines that also contain a short-lived firefly luciferase reporter for the expression of the target endogenous connective tissue growth factor (ctgf ) gene allowed us to quantitatively link nuclear accumulation of SMADs upon TGF-Î² stimulation to the target gene expression in real-time single cell measurements. Time- lapse luminescence microscopy and image analysis with the custom developed CAST (Cell Automated Segmentation and Tracking platform) platform provided quantitative single-cell data to link the upstream transcription factor profile to its target gene activity. The data revealed weak single-cell correlations between translocation level and the target gene expression response which suggests a mechanism in which the translocation of SMADs initiates the transcriptional response but affects the response amplitude minimally. However, SMAD4 expression levels influenced the target gene response dynamics such that cells with high SMAD4 abundance favored to respond in a more sustained and even oscillatory manner. This suggests a mechanism consisting of a dynamic interplay between the transcriptional activators and feedback mechanisms in TGF-Î² signaling. We explored further the effect of different factors such as ligand concentration, ligand type on both signaling and target gene response. Taken together, this study proposes an experimental and quantitative framework that dissect mechanisms underlying transcriptional response to stimulus.

EPFL2018

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

A gateway-compatible yeast one-hybrid system

Bart Deplancke

Since the advent of microarrays, vast amounts of gene expression data have been generated. However, these microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets. Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter. To understand the mechanisms underlying differential gene expression, it is necessary to identify physical interactions between regulatory TFs and their target genes. We developed a Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments. We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system. We could detect approximately 40% of previously reported TF-promoter interactions. By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter. We recapitulated several of the Y1H-based protein-DNA interactions using luciferase reporter assays in mammalian cells. Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.