A defining feature of complex multicellular organisms is their ability to generate multiple cell types with specific phenotypes and behaviors. During development, this process must be precisely coordinated through the generation of cell-specific transcriptional programs. In the adult organism, specific transcriptional programs also influence tissue homeostasis by controlling processes such as cell turnover, behavior and physiological status. Thus, a critical question in both development and physiology is how transcription is controlled. We focus on the genome-wide onset of transcription in zebrafish, with the aim to understand how the transcriptional machinery and chromatin template are brought together in time and space to robustly regulate transcription in hundreds of blastomeres during genome activation.
We analyze transcriptional regulation quantitatively (quantification of repressors, activators, number of cells at genome activation, transcripts) and at high resolution (imaging transcripts, chromatin architecture, and transcriptional machinery at high resolution) in the context of the embryo. This allows us, especially in collaboration with physicists, to address questions about the quantitative relationship between activators and repressors, the timing of transcription initiation during development, synchrony between cells, and the function and establishment of nuclear architecture.
1. The interaction between transcriptional machinery and chromatin template
Upon fertilization, the genome of animal embryos is transcriptionally inactive until the controlled onset of transcription during the maternal-to-zygotic transition. A key factor in regulating this transition is the accessibility of the genome for DNA binding proteins. Thus, the onset of transcription during genome activation provides a perfect system to study the interaction of the transcriptional machinery with the chromatin template.
We have recently found that a competition between histones and transcription complex assembly regulates the onset of transcription during embryogenesis (Joseph et al., eLife 2017 PMID: 28425915; Palfy et al., COGD 2017 PMID: 28088031). Initially, there is a large excess of histones stockpiled in the embryo. The manipulation of histone levels in vivo revealed that this excess dictates the timing of transcription onset in zebrafish embryos. Our quantitative mass spectrometry approach (Kumar et al., under review at Molecular & Cellular Proteomics) in combination with subcellular histone distribution analysis showed that the time of genome activation coincides with a reduction in the nuclear concentration of non-DNA bound histones. This provides opportunity for critical transcription factors (such as Pou5f3 and Sox19b) to bind to DNA and initiate transcription. Following this argument, the concentration of transcription factors also affects the time of transcription. Together, our results demonstrate that the relative levels of histones and transcription factors determine the timing of zygotic genome activation by competing for access to DNA. This is, to our knowledge, the first example of a developmental transition in which competition for DNA binding between histones and transcription factors plays an important role in transcriptional regulation.
We are currently
- Working on a theoretical model that describes the temporal regulation of transcription in embryos (in collaboration with Vasily Zaburdaev, MPI-PKS).
- Analyzing the kinetics of transcription activation.
- Developing a cell free assay for transcription, to further analyze the molecular mechanism of competition.
- Analyzing when and where in the genome competition occurs.
Moving forward, we will
- Address the generality of the competition model, both in other species, as well as during other important developmental transitions.
Taken together, these studies will reveal how the transcriptional machinery competes with chromatin assembly to regulate transcription, not only during genome activation but potentially also during differentiation and reprogramming.
2. The role of chromatin structure and nuclear architecture in genome activation
Chromatin regulates the accessibility of the genome for DNA binding proteins. Changes in chromatin structure and nuclear organization are thus critical to understanding how regions of the genome become transcriptionally competent. We have previously shown that dramatic changes in chromatin structure accompany the onset of zygotic gene expression (Vastenhouw et al., Nature 2010 PMID: 20336069; Zhang et al., GR 2014 PMID: 24285721). Thus, the onset of transcription during genome activation provides a powerful system to study the relationship between chromatin structure and transcriptional activity.
We have recently found that transcriptional activity and accumulation of the produced RNA transcripts locally remodel DNA. We developed an in vitro system for genome activation using dissociated blastomeres to image the nucleus at high resolution as the genome starts to engage in transcription. Using super-resolution and live cell microscopy of DNA, RNA, and transcriptional activity in embryonic zebrafish cells, we find that nuclear RNA accumulates around transcription sites, in the process displacing not transcribed DNA but retaining transcribed DNA. Using a physical model, we identified two essential mechanisms of this process: segregation of RNA-protein complexes from DNA, and tethering of RNA-protein complexes to transcribed DNA (Hilbert et al., in preparation). The microscopic mechanisms and effective patterns place this model in a new class of physical systems, active microemulsions. Our findings explain how transcriptional activity and RNA accumulation shape local microenvironments, which serve as basic units of euchromatin organization throughout the cell nucleus. We hypothesize that such microenvironments might function as hubs for genome organization and transcriptional activity.
We are currently
- Further exploring the relationship between chromatin structure and transcription during early embryogenesis, by characterizing the changes in DNA accessibility during genome activation using a ATAC-seq (assay for transposase-accessible chromatin coupled to high-throughput sequencing) and how this depends on specific transcription factors.
Moving forward, we will
- Analyze the functional relevance of nuclear organization on transcriptional output.
3. The coordination of gene expression between cells
Transcription is often stochastic, resulting in significant variation in transcript levels between cells in a population. Developmental processes, like gastrulation, however, have been suggested to rely on uniform gene expression patterns. This raises the question how embryos deal with the stochastic nature of transcription. Early development of zebrafish embryos provides an ideal experimental platform for elucidating the establishment of gene expression patterns required to support development. The onset of transcription during zygotic genome activation is followed by gastrulation after three cell cycles, providing a window of opportunity to dissect how uniform expression patterns are established.
We developed a method to quantify gene expression levels during zebrafish embryogenesis at single cell and single molecule resolution (Stapel et al., 2017 PMID: 26700682). Using this method, we find that genes are stochastically activated, as a consequence of (i) genes acquiring transcriptional competence at different times in different cells, (ii) differences in cell cycle stage between cells (extrinsic noise), and (iii) the stochastic nature of transcription itself (intrinsic noise). Stochastic transcription initially leads to large cell-to-cell differences in transcript levels but this variability is reduced over time. The acquisition of transcriptional competence is a one-time event affecting transcript variability only when transcription starts. The effects of extrinsic noise caused by cell cycle heterogeneity are reduced by continuous cell division and cell cycle lengthening. Finally, the effects of intrinsic noise are reduced due to the accumulation of multiple transcription events in each cell. Thus, the stochastic nature of transcription does not present a problem for the embryo because temporal averaging of gene expression noise prevents the propagation of large differences in transcript levels (Stapel et al, in revision).
Moving forward, we will
- investigate the relationship between nuclear architecture and transcriptional stochasticity.
Our ultimate goal is to generate a uniform model for transcriptional regulation. We attempt to understand how all of the different variables that influence the decision to transcribe a gene or not come together to generate complex transcriptional programs. We use the genome wide onset of transcription as a way into this question and will progressively work our way into cell-specific programs, until we ultimately delineate an overall model of transcriptional control.