Wednesday, August 10, 2022
8/10/2022
PROJECT SUMMARY Regulatory T cells (Treg) play a critical role in maintaining immune system homeostasis and preventing autoimmunity and immunopathology. Defective Treg function is linked to multiple autoimmune diseases including type 1 diabetes and multiple sclerosis. On the other hand, enrichment of Treg cells within tumors is thought to be a barrier to effective anti-tumor immune responses. The development and maintenance of the Treg cell lineage are dependent on the transcription factor Foxp3, as loss of function mutations lead to severe lymphoproliferative disease in mice and humans. Thus, understanding the mechanisms that govern Foxp3 induction and stability may lead to the development of novel therapies for autoimmune disease and cancer. Dr. Zheng and colleagues recently developed a system to perform genome-wide CRISPR/Cas9 knockout screens to identify Foxp3 regulators in mouse Tregs. The unbiased screen results not only confirmed a number of known Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis of newly identified Foxp3 regulators revealed eIF5A itself and two genes involved in its hypusination, indicating a previously unknown role for hypusinated eIF5A (eIF5AH) in controlling Foxp3 expression and Treg function. Hypusination is a post-translational modification of lysine residue by the action of two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) using spermidine as a substrate. A conserved lysine residue in eIF5A is the only known hypusination target site in eukaryotes. Upon hypusination, eIF5AH functions as a facilitator that promotes mRNA translation initiation, elongation and termination. Although eIF5AH is known for regulating cell growth, how hypusination pathway maintains cell identity and cell type specific function is unclear. The unbiased genome-wide Treg screen identified eIF5A, DHPS and DOHH as negative regulators of Foxp3 expression. Deletion of each of these genes by CRISPR knockdown leads to significantly increase of Foxp3 expression, and surprisingly, reduced Treg suppressor function. This unusual decoupling of high Foxp3 expression with increased suppressor function suggests that hypusination pathway adds a new layer of regulatory mechanism controlling Treg function. The overall objective of this study is to define the role of hypusinated eIF5A in Foxp3 expression and Treg function. This goal will be accomplished by elucidating mechanistically on how eIF5AH regulates expression of Foxp3 and other signature genes in Tregs (Aim 1), and exploring the in vivo consequence of blocking hypusination in conditional knockout mouse models (Aim 2). The outcomes of the proposed studies are expected to fundamentally advance the understanding on how hypusinated eIF5A regulates Foxp3 expression and Treg function. The outcomes of this research could provide evidence to support targeting hypusination pathway to enhance anti-tumor immune response.
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