Tools for reversible short-term degradation of TCF-1 to address its molecular functions

TCF-1短期可逆降解工具研究其分子功能 摘要 在T细胞发育过程中，T细胞特异性DNA结合蛋白TCF-1执行多个阶段和 特定的表观遗传功能。Tcf-1与增强和减少染色质有关 可及性和基因表达取决于它结合的区域和其他调节子的近端结合。 在TCF-1如何利用其阶段和特定背景的功能方面，我们的知识存在差距。TCF-1是 对于启动祖细胞对T细胞谱系的承诺是必不可少的，它调节表观遗传学 以及发育中的T细胞在几乎每个发育转变和T细胞谱系中的转录图谱。 传统上，TCF-1被认为是典型的WNT信号通路的下游效应器，结合 由β-连环蛋白响应WNT的激活而发挥转录激活剂的作用。然而，TCF-1有两个主要功能 只有全长蛋白(Tcf-1p45)能与β-连环蛋白相互作用的异构体，而短的异构体(Tcf-1p45)能与Tcf-连环蛋白相互作用。 1p38)不能。因此，TCF-1可以在WNT级联内运行或独立于WNT级联运行。的直接约束 Tcf-1的保守基序可以弯曲DNA螺旋，并有可能改变3D染色质的构象 全基因组对染色质景观、基因表达和细胞的未知后果 发展。目前的证据表明，TCF-1的功能结果取决于 与其共同招募到DNA的调节器以及TCF-1基序在 招聘网站。根据这一证据，我们推测TCF-1通过结合调节T细胞的发育 在离散调控复合体的背景下选择性地塑造表观遗传格局的DNA， 3D染色质构象和发育中T细胞的基因表达。以最佳方式解决这一问题 长期缺乏TCF-1导致的没有混淆发育缺陷的假说需要 T细胞特定阶段选择性TCF-1亚型蛋白水平的可诱导性短期调控 发展。因此，要在时间和空间上研究TCF-1的分子和表观遗传功能， 建议建立和验证三种动物模型，当组合在一起时，允许在体内诱导 TCF-1亚型的短期降解。一个模型将有条件地表达Oryza的优化版本 Sativa TIR1蛋白(OsTIR1(F74G))，能够在体内精确地在体内降解任何标记的女佣 蛋白质以一种组织和发育阶段特有的方式。另外两个模型将生成标记为maid的 Tcf-1p45或所有Tcf-1亚型这项研究不仅将为TCF1长期存在的问题提供至关重要的见解 功能和其亚型之间的分工，但也将提供新的有效的小鼠模型 为更广泛的社区提供资源。

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 β-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 β-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.