Periodic Reporting for period 2 - Hotspot (Understanding gene regulation in nuclear space)
Periodo di rendicontazione: 2023-01-01 al 2024-06-30
There is accumulating evidence for a new gene expression paradigm, in which genes and transcription factors cooperate to create specialized nuclear hotspots (from here on called transcription bodies) optimized for transcription. How transcription bodies form, and how they impact transcription, however, is unclear. This is mostly because mechanistic and functional approaches have been limited by the large number of transcription bodies per nucleus, their small size, and their transient nature.
We take advantage of two transcription bodies that precede all other transcription during zebrafish embryogenesis. The hotspots are large, isolated, and relatively long-lived, and we can interfere with their formation specifically. They therefore provide an excellent opportunity to study transcription bodies. The stereotyped activation of transcription during embryogenesis further provides the opportunity to determine the effect of hotspots on gene expression.
In Aim 1, we will determine what triggers transcription body formation, identify the components of hotspots and the order in which they come together, and determine the enrichment of components in hotspots compared to nucleoplasm.
In Aim 2, we turn our attention to the genes in these hotspots. We will develop a method to visualize single copy genes live and use this to determine how genes come together in transcription hotspots to be activated.
Together, the work we propose will reveal how stable gene expression programs can emerge from the self-organizing interactions between chromatin, transcription factors and emergent properties of transcribed sites.
We take advantage of two transcription bodies that precede all other transcription during zebrafish embryogenesis. The hotspots are large, isolated, and relatively long-lived, and we can interfere with their formation specifically. They therefore provide an excellent opportunity to study transcription bodies. The stereotyped activation of transcription during embryogenesis further provides the opportunity to determine the effect of hotspots on gene expression.
In Aim 1, we will determine what triggers transcription body formation, identify the components of hotspots and the order in which they come together, and determine the enrichment of components in hotspots compared to nucleoplasm.
In Aim 2, we turn our attention to the genes in these hotspots. We will develop a method to visualize single copy genes live and use this to determine how genes come together in transcription hotspots to be activated.
Together, the work we propose will reveal how stable gene expression programs can emerge from the self-organizing interactions between chromatin, transcription factors and emergent properties of transcribed sites.
Related to Aim 1, -
We have used these two transcription bodies to characterize the dynamics of transcription body formation (Kuznetsova et al., 2023). We discovered that once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we found that Nanog and Sox19b cluster prior to transcription. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies.
Related to Aim 2, -
We have disrupted the formation of two prominent transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analyzed the effect on gene expression using enriched SLAM-Seq and live-cell imaging (Ugolini et al., accepted for publication in Nature Cell Biology). We found that the disruption of transcription bodies results in the upregulation of genes that already have accessible chromatin and are poised to be transcribed when the two transcription bodies are present. Live-cell imaging suggests that the disruption of these transcription bodies enables poised genes to be transcribed in ectopic transcription bodies. Our results suggest that transcription bodies regulate transcription genome-wide by sequestering components of the transcriptional machinery.
We have used these two transcription bodies to characterize the dynamics of transcription body formation (Kuznetsova et al., 2023). We discovered that once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we found that Nanog and Sox19b cluster prior to transcription. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies.
Related to Aim 2, -
We have disrupted the formation of two prominent transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analyzed the effect on gene expression using enriched SLAM-Seq and live-cell imaging (Ugolini et al., accepted for publication in Nature Cell Biology). We found that the disruption of transcription bodies results in the upregulation of genes that already have accessible chromatin and are poised to be transcribed when the two transcription bodies are present. Live-cell imaging suggests that the disruption of these transcription bodies enables poised genes to be transcribed in ectopic transcription bodies. Our results suggest that transcription bodies regulate transcription genome-wide by sequestering components of the transcriptional machinery.
Progress beyond the state of the art is described above.
We expect to
- Identify in an unbiased manner, the components of transcription bodies (Aim 1.2)
- Visualize these components and interfere with them to determine how they contribute to transcription body formation (Aim 1.2)
- Characterize protein domains and concentrations required for clustering (Aim 1.3)
- Investigate the concentration of transcriptional machinery in transcription bodies and nucleoplasm (Aim 1.3)
- Visualize genes live as they move through nuclear space.
We expect to
- Identify in an unbiased manner, the components of transcription bodies (Aim 1.2)
- Visualize these components and interfere with them to determine how they contribute to transcription body formation (Aim 1.2)
- Characterize protein domains and concentrations required for clustering (Aim 1.3)
- Investigate the concentration of transcriptional machinery in transcription bodies and nucleoplasm (Aim 1.3)
- Visualize genes live as they move through nuclear space.