Year: 2020
Type: dissertation
Abstract: Developing T-cells require the coordinated function of various transcription factors to promote T-lineage progression, among which is the E-protein HeLa E-box Binding (HEB) factor involved in promoting T-lineage specification and commitment. HEB-deficient mice have altered gene expression, changes in proliferation in the early double-negative (DN) stages, and blocks in T-cell development at the CD8+ Immature Single Positive (ISP8) stage. Although previous studies highlighted the importance of HEB in T-cell development, they failed to distinguish between the HEB isoforms HEBCan (Canonical) and HEBAlt (Alternative) which are produced by different transcription start sites. I set out to identify the functional differences between the HEB factors by investigating specifically the function of HEBAlt in the context of T-cell development. I began by investigating whether the Alt-domain, the defining feature of HEBAlt, is required for HEBAlt transcriptional activity. My findings demonstrated that phosphorylation of a tyrosine (Y)-rich YYY motif is required to activate HEBAlt, and that this phosphorylation may be dependent on Janus kinases, as a result of interleukin-7-receptor signaling within the thymus. Phosphorylation of HEBAlt does not affect subcellular localization and the activity of other E-proteins, however it reduces protein degradation. HEBAlt phosphorylation at the YYY-motif results in greater resistance to Inhibitor of DNA-binding protein-mediated inhibition and comparable transcriptional activity to other E-proteins involved in T-cell development. Phosphorylated HEBAlt also appears to be required for the regulation of a pre-T Cell Receptor- enhancer. In addition, we generated HEBAlt-transgenic and HEBAlt-deficient mice. Our analyses showed that HEBAlt-transgenic mice have reduced proportions of ISP8 cells, opposite to HEB-deficient mice, suggesting HEBAlt may promote developmental progression at the ISP8-to-CD4+CD8+ Double Positive (DP) stages. In vitro T-cell differentiation of HEBAlt-transgenic progenitors have reduced ISP8 cell and greater DP cell proportions, further supporting the role for HEBAlt promoting faster ISP8-to-DP transition. Interestingly, HEBAlt-deficient mice appeared to have no altered proportions in their ISP8 compartments, possibly due to the presence of HEBCan and other E-proteins that may compensate for HEBAlt absence. Overall, our results suggest that HEBAlt function requires phosphorylation at the YYY-motif to be activated, and that HEBAlt may promote the transition of ISP8-to-DP cells in T-cell development.
Source: TSpace (University of Toronto)
Author: Kogulan Yoganathan
Language: English
Topic: T-cell and B-cell Immunology
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
Funder: Sunnybrook Research Institute
OpenAlex ID: W3114222301