Cellular and molecular analysis of thymus organogenesis and maintenance
Description: 1. Background and rationale of the projectThe thymus provides the physiological microenvironment for the development of T lymphocytes and is therefore essential for the successful establishment and maintenance of the immune system's capacity to distinguish between vital self and injurious non-self. This fundamental competence is primarily instructed by thymic epithelial cells (TECs) whose differentiation and functions are still incompletely understood albeit critically controlled by the master regulator FOXN1. We have identified the CREB-binding protein (CBP) as an essential co-factor for FOXN1-controlled transcription and shown that spontaneously occurring mutations of human FOXN1 precluding a physical interaction with CBP cause athymia and severe T cell lymphopenia. Moreover, the spontaneous heterozygosity of CBP in humans (Rubinstein-Taybi Syndrome) results thymus hypoplasia/aplasia and the engineered loss of CBP expression in mouse TEC causes thymic thymic hypoplasia in mice with substantial changes in the composition of individual TEC states. We therefore seek to elucidate at a molecular, cellular, and organ level how CBP cooperates with FOXN1 and in what way this interaction controls normal TEC subset differentiation and function.2. Working hypothesesThe proposed research is based on the demonstrated requirement of FOXN1 to cooperate with CBP to effect its role as a transcription factor. We hypothesise a physical binding to be required between the two molecules via FOXN1’s intrinsically disordered regions. As a result, CBP post-transcriptionally modifies FOXN1, alters histone marks at target sites, enables local chromatin accessibility and shapes the 3D genome architecture, to collectively control TEC development and function. 3. Overall objectives and specific aims The project’s overall objective is to define the role of CBP and its interaction with FOXN1 to create and maintain a normal TEC scaffold supporting regular thymopoiesis. Specifically, three aims will be experimentally addressed:•Aim 1: Structure-function analysis of FOXN1’s interactions with the co-factor CBP, its post-translational modifi-cations and epigenetic consequences.•Aim 2: The in vivo impact of a TEC-targeted loss of CBP expression on the cells’ developmental potential, thymo-poietic function, and gene expression profiles and chromatin accessibility in separate TEC subtypes and states.•Aim 3: Definition of the role of CBP and FOXN1 in the 3D chromatin organisation of distinct TEC subpopulations. 4. Experimental Design and Methods The proposed research programme builds on our recently published work and extensive data from preliminary experiments defining the role of CBP and its cooperativity with FOXN1. The chosen experimental design allows to interrogate CBP’s molecular role in vitro which will be complemented and verified in vivo investigating animals with a TEC-targeted, yet graded loss of CBP expression. State-of- the-art methods will be employed for the proposed studies, including cytometry-based deep phenotyping, transcriptomic investigations at both population level and single cell resolution, high-parameter single cell spatial imaging, and studies of the 3D chromatin organisation and locus-specific chromatin accessibilities in individual TEC subtypes. The combination of these individual methods comprehensively informs not only on CBP’s pivotal role in TEC biology but also provides insight into the molecules wider function in chromatin organisation.5. Expected results and their impact for the field and beyond.The thymus is essential for the formation and maintenance of an effective adaptive immune system. The proposed work will provide unique molecular details regarding the role of CBP in chromatin architecture and accessibility, and its physical co-operativity with FOXN1 effecting post-translational modifications and the control of gene transcription. Collectively, insight will be gained from the proposed work how normal TEC lineage differentiation, function and maintenance depend on the availability of CBP. In addition, these studies will provide at single cell resolution CBP’s general role in shaping distinct 3D genome architectures that may closely relate to a cells’ gene expression profiles and functions.
Amount: CHF 880,000
Funding type: research
Funder scheme: Project funding
Start date: 2023-07-01
End date: 2027-02-28
Investigator: Hollander, Georg
Institution: University of Basel – BS
Funded outputs count: 1
Funder award ID: 215113
OpenAlex ID: G4846973657