Understanding the role of:
1. DNA binding
3. Insulator proteins
4. Polycomb components in the establishment of chromatin architecture during neuronal differentiation.
The genome of higher eukaryotes is organised in a hierarchy of structures — From chromatin loops, to topologically associating domains (TADs), to genome compartments (GCs), to chromosome territories (CTs) that are spatially co-organised in the 3D space of the cell nucleus. At each level, chromatin architecture is regulated and impinges on genome functions, such as gene expression, DNA replication and repair.
Recent studies, including work in the participant’s labs, have identified players that define loops, TADs and GCs, but their mechanisms of action are not understood. In particular, the boundaries of TADs can involve transcribed genes, but shows that the act of transcription per se is not sufficient in order to drive the formation of a TAD. Furthermore, CTCF can form TAD boundaries, but little is known of its mechanism of action. Drosophila also has a CTCF protein that binds the same sequence motifs, but does not suffice to specify TAD boundaries in that species.
Finally, gene silencing components of the Polycomb group were shown to induce the formation of extremely long-range interactions (ELRI) over tens of Mb of linear distance in ES cells, but many of these are disrupted in neurons and it is not known whether this is important for differentiation. Dissecting these aspects of genome regulation, should have profound implications to understanding differentiation in normal cells and diseases (cancer), where nuclear architecture is a major alteration frequently observed but not understood.
ESR11 will drive DNA-binding proteins to bind in a genomic region where there is normally no such binding, or prevent them from binding when they should. Studying the effect of DNA binding perturbation on the 3D domain architecture, by separating out the effects of DNA-binding from those of transcriptional induction.
In addition, ESR11 will dissect the function of mouse vs Drosophila CTCF. Replacing mouse CTCF with it’s fly counterpart and study cell viability and 3D genome function. Vice versa, she/he will express the mouse protein into flies, dissecting them further by making partial swaps of only the DNA binding domain or of portion of its intrinsically disordered portion.
Hi-C and microscopy will identify protein portions involved in the species-specific functions for this gene. Finally, ESR11 will analyse the specific function of one PcG protein group, including the PHC1, PHC2 and PHC3 components of the PRC1 complex. The expression of these genes varies during neuronal differentiation, concomitantly with the strong loss of ELRIs. Identifying which of these proteins can fulfil this function along with the study of its mechanisms of action and the consequence of ELRIs in neuronal differentiation.
Thanks to the secondments, ESR11 will have the possibility to optimise the streamline of the project, performing biochemical studies and physical modelling studies labs at CRG- CNAG.
Increased understanding of determinants of 3D genome organisation and cell differentiation.
CRG, Spain (3 months):
Production of specific antibodies and proteomics analysis of ES cells expressing FLAG-HA tagged PHC Knock-in genes.
CNAG-CRG, Spain (3 months):
Physical modeling of the 3D genome architecture in wild type and engineered cells.
Milner Therapeutics Institute, United Kingdom (1 month):
Learning or testing drug development based on results.
Enrolment in doctoral programs
Doctoral School of the University of Montpellier
Bonev, B. et al. Multiscale 3D Genome Rewiring during Mouse Neural Development. Cell 171, 557-572 e524, doi:10.1016/j.cell.2017.09.043 (2017).
Ogiyama, Y., Schuettengruber, B., Papadopoulos, G. L., Chang, J. M. & Cavalli, G. Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development. Mol Cell 71, 73-88 e75, doi:10.1016/j.molcel.2018.05.032 (2018).
Schuettengruber, B., Bourbon, H. M., Di Croce, L. & Cavalli, G. Genome Regulation by Polycomb and Trithorax: 70 Years and Counting. Cell 171, 34-57, doi:10.1016/j.cell.2017.08.002 (2017).