This confederation of projects is headed by Mark I. Greene who has been involved in the study of immune regulation and phenotype reversal for over 30 years. The goals of this highly integrated program project are to develop understanding of how the FoxpS proteins exert their biological effects and through this knowledge to develop translationally relevant therapeutics that disable or activate the Foxp3 complex. Although the theme of Foxp3 function resonates in each project, a variety of distinct technologies are actually employed to develop insights into how Foxp3 complexes operate and how to manipulate them, leading to a highly interactive group of experiments that should lead to therapeutics that will reach the clinic within the time frame of this program project. The long-term goals are reiterated in every project and all projects share recurrent themes of Treg phenotype manipulation. The studies to date have already lead to a rational therapeutic for autoimmune conditions that is entering a preliminary clinical trial at the NIH. The goal of Greene's project is to provide basic biochemical information of how the Foxp3 complex binds to chromatin in human cells. This information will be helpful in Andrew Wells's study of mouse chromatin - Foxp3 interactions and will be useful in the creation of transgenic and mutant mice that will help the Hancock project and the Wells project. Human and mouse Foxp3 complexes appear to have differences although both form large ensembles. The intent of Project 1 is to identify individual residues that are acetylated and phosphorylated and subdomains that mediate interactions with other repressive components. This information will provide a framework for Andrew Wells to examine chromatin remodeling events in the mouse and for Wayne Hancock to examine functional relevance in in vivo models. PROJECT 1: Foxp3 and immune regulation (Greene, M) PROJECT 1 DESCRIPTION (provided by applicant): Maintenance of unresponsiveness to self-antigens is essential for the prevention of autoimmunity but is an incompletely understood process. Our studies will focus on certain biochemical features of regulatory T cells (Treg) and how the Foxp3 complex mediates its repressive effects. While it is clear that mutations in human FOXP3 predispose individuals to human autoimmune conditions, it is unclear why the mutant protein fails to function as a transcriptional regulator. There is also limited detail of how FOXP3 itself interacts with the transcriptional machinery and which components of the FOXP3 ensembles exert phenotypic changes to render cells able to mediate suppression. Our proposed studies focus on the biochemistry of FOXP3 complexes, as well as in vivo models to examine modification of Treg function through rational biochemical alteration of the FOXP3 ensemble. The studies provide compelling evidence that a complex of specific histone acetyl transferases and histone deacetylases (HDAC) associate with FOXP3 to create a transcriptional represser. FOXP3 becomes acetylated and phosphorylated and is then able to mediate its activities. The essential areas of the grant focus on the signaling effects that lead to post translational changes and functions of the Foxp3 ensemble. A dominant theme that emerges is that Foxp3 is acted on by enzymes that modify its activity and stability and interactions with chromatin which lead to alterations in the actions of Treg in vitro and in vivo. A rational therapeutic emerges from these studies employing HDAC inhibitors to increase Treg acetylation modifications at specific lysine residues and thereby increase Treg function to ameliorate autoimmunity. The goal of Project 1 is to provide basic biochemical information of how the Foxp3 complex binds to chromatin in human cells. This information will be helpful in Andrew Wells's study of mouse chromatin- FoxpS interactions and will be useful in the creation of transgenic and mutant mice that will help the Hancock project and the Wells project. Human and mouse Foxp3 complexes appear to have differences. The intent of this project is to identify individual residues that are acetylated and phosphorylated and subdomains that mediate interactions with other repressive components. This information will provide a framework for Andrew Wells to examine chromatin remodeling events in the mouse and for Wayne Hancock to examine functional relevance in in vivo models.
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