Architectural Rearrangements at the Xist locus during the onset of X-chromosome inactivation
Developmental genes are often regulated by long-range enhancer elements. To activate gene expression, these elements must come into close physical proximity of the gene promoter. Enhancer-promoter contacts mostly occur within topologically-associating domains (TADs), which are submegabase regions, where chromatin preferentially interacts. TAD positions are mostly invariant across cell types, but intra-TAD contacts often accompany changes in gene expression. Several mechanisms have been proposed to drive such intra-TAD rewiring, but how they cooperate to precisely tune gene expression remains poorly understood. In the proposed project, we will systematically test several alternative hypotheses of how intra-TAD rewiring is controlled and how these architectural changes shape transcriptional output. We will use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. We will make use of a system in murine embryonic stem cells (TX XXΔXic/XO) we have recently developed, which allows high-resolution profiling of the Xist locus at the inactive X, where Xist is expressed, and at the active X, where Xist is silent. Using this cell model we have discovered structural rearrangements during initial Xist upregulation within the TAD that harbors the Xist promoter. In the proposed project, we will build on our extensive characterization of the TX XXΔXic/XO model, with respect to chromatin modifications, transcription and chromosome conformation. We will complement the existing data with factors that have been proposed to govern 3-dimensional genome structure and with high-resolution quantification of 3D contacts. We will then use computational modelling and experimental perturbations to test several alternative hypotheses of how 3D rewiring at the Xist locus might be controlled. Specifically, we will perform polymer simulations of cohesin-mediated loop extrusion, assuming (1) differential binding of CTCF, which can halt extrusion, (2) preferential cohesin loading at activated enhancers, or (3) lncRNA transcription as extrusion barrier. All three hypotheses will also be tested by (epi)genomic perturbations using the CRISPR/Cas9 system. In addition, we will use the high-resolution contact mapping data for unbiased identification of proteins bound at interacting genomic sites, to potentially develop additional hypothesis of how 3D rewiring might be regulated. Finally, we will assess the functional consequences of 3D rewiring, by quantifying transcriptional output in response to perturbations of 3D contacts. By combining a series of state-of-the-art experimental and computational approaches we will thus dissect how dynamic changes in genome architecture are regulated during an essential developmental process. The results will be relevant beyond the X inactivation field, as we will, for the first time, precisely dissect the relative contributions of different mechanisms to the dynamics of chromatin contacts at a specific locus.
Dr. Edda Schulz,Berlin
Max-Planck-Institut für molekulare Genetik (MPIMG)