Tools for reversible short-term degradation of TCF-1 to address its molecular functions
Abstract
During T cell development, the T cell-specific DNA binding protein TCF-1 performs multiple stage and
lineagespecific epigenetic functions. TCF-1 has been associated with both enhancing and reducing chromatin
accessibility and gene expression depending on the region it binds to and the proximal binding of other regulators.
There is a gap in our knowledge in how TCF-1 leverages its stage and context-specific functions. TCF-1 is
essential for initiating the commitment of progenitor cells to the T-cell lineage and it modulates the epigenetic
and transcription profiles of developing T-cells at almost every developmental transition and T-cell lineage.
Classically TCF-1 has been viewed as a downstream effector of the canonical WNT signaling pathway, bound
by ß-catenin in response to WNT activation to act as a transcriptional activator. However, TCF-1 has two main
isoforms of which only the full-length protein (TCF-1p45) can interact with ß-catenin, while the short isoform (TCF-
1p38) cannot. Therefore, TCF-1 can function within or independently of the WNT cascade. The direct binding of
TCF-1 to its conserved motif bends the DNA helix and has the potential to alter the 3D chromatin conformation
genome-wide with unknown consequences on the chromatin landscape, gene expression, and cellular
development. Current evidence suggests that the functional outcomes of TCF-1 depends on the group of
regulators with which it is co-recruited to DNA and on the presence or absence of the TCF-1 motif at the
recruitment sites. Based on this evidence we postulate that TCF-1 regulates T cell development by binding
to DNA in the context of discrete regulatory complexes to selectively shape the epigenetic landscape,
3D chromatin conformation, and gene expression of developing T cells. Optimally addressing this
hypothesis without confounding developmental defects resulting from long-term TCF-1 deficiency requires
inducible short-term manipulation of the protein levels of selective TCF-1 isoforms at specific stages of T cell
development. Therefore, to study the molecular and epigenetic functions of TCF-1 in time and space, it is
proposed to generate and validate three animal models that when combined allows the inducible, in vivo
shortterm degradation of TCF-1 isoforms. One model will conditionally express the optimized version of the Oryza
sativa TIR1 protein (OsTIR1(F74G), which enables precise temporal in vivo degradation of any mAID tagged
protein in a tissue and developmental stage specific manner. The other two models will generate mAID tagged
TCF-1p45 or all TCF-1 isoforms This research will not only offer crucial insight into longstanding questions of TCF1
functionality and the division of labor between its isoforms but will also provide novel validated mouse models as
a resource to the wider community.