The packaging of genomic information into chromatin and its organization in three-dimensional (3D) space affects all aspects of DNA metabolism, including transcription, replication, repair, and recombination. Indeed, considerable crosstalk exists between mechanisms controlling gene expression and genome conformation, both of which rely on dynamic changes in composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain largely unknown. Antigen receptor loci are excellent models to study these mechanisms because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. For example, upon commitment to the earliest stage of T cell development, the Tcrb antigen receptor locus folds into a compact conformation that promotes long-range recombination between V? gene segments (Trbvs) and their D?J? targets. The applicant has discovered a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for Tcrb diversification. Thymocyte-specific association between distal Trbv segments and the highly expressed D?J? clusters, termed the recombination center (RC), relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Trbv array to the RC. The second is a boundary element (BE) that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts distal Trbv contact and significantly alters Tcrb repertoires. These findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers. Importantly, the discovery prompts a more general hypothesis: that the long-range tethering function of at least some CTCF sites is disarmed when they are forced to become chromatin boundaries. If correct, our findings would establish a new paradigm for how genome architecture is regulated to coordinate patterns of gene expression. In this exploratory project, we will take the first essential steps toward proving our hypothesis, using Tcrb as a model. For this purpose, experiments are proposed to (i) rigorously define the Tcrb boundary element that protects the tethering CTCF site from hyperactive chromatin, (ii) prove that the CTCF site serves as the primary tether for distal Trbv segments via its deletion in mice, and (iii) independently test whether acquisition of boundary function by the CTCF site consistently disrupts long-range folding of Trbvs into the active RC. Resolution of these issues in the topological regulation of Tcrb will lend important insights into the menu of mechanisms that can be deployed to control gene expression programs in response to developmental cues or physiologic agonists.
The packaging of our genome in cell nuclei and its 3D spatial organization are key determinants of gene function; changes in these 'epigenetic' programs are at the heart of aging and disease. In addition, epigenetic control is essential for the proper generation of antigen receptor genes; defects in which can cause chromosomal translocations leading to lymphoid tumors. We propose studies to address how precursor T lymphocytes control long-range changes in the spatial organization of T cell receptor beta and other genomic loci to ensure proper gene expression programs that guide cellular development.
| Huang, Yue; Koues, Olivia I; Zhao, Jiang-Yang et al. (2017) cis-Regulatory Circuits Regulating NEK6 Kinase Overexpression in Transformed B Cells Are Super-Enhancer Independent. Cell Rep 18:2918-2931 |
| Zhao, Jiang-Yang; Osipovich, Oleg; Koues, Olivia I et al. (2017) Activation of Mouse Tcrb: Uncoupling RUNX1 Function from Its Cooperative Binding with ETS1. J Immunol 199:1131-1141 |
