Zhimei Gao

Identification of the E3 Deubiquitinase Ubiquitin-specific Peptidase 21 (USP21) as a Positive Regulator of the Transcription Factor GATA3 *

Background: GATA3 is regulated both transcriptionally and post-translationally. GATA3 is important for the function of FOXP3+ Treg cells. Results: USP21 prevents the ubiquitination and degradation of GATA3. Conclusion: USP21 is a positive regulator of GATA3 expression. Significance: The identification of a molecular pathway where USP21 positively controls GATA3 expression at the post-translational level reveals USP21 as a potential drug target to manipulate the function of T cells. The expression of the transcription factor GATA3 in FOXP3+ regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.

PIM1 kinase phosphorylates the human transcription factor FOXP3 at serine 422 to negatively regulate its activity under inflammation.

Background: FOXP3 is a key transcription factor for the development and function of Tregs. Results: PIM1-mediated phosphorylation of FOXP3 at serine 422 decreased its DNA binding activity. Conclusion: PIM1 negatively regulates FOXP3-mediated transcriptional regulation and the suppressive activity of Tregs. Significance: PIM1 is a newly identified negative regulator of the immunosuppressive activity of Tregs. Previous reports have suggested that human CD4+ CD25hiFOXP3+ T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser422. Our data also suggest that phosphorylation of FOXP3 at the Ser418 site could prevent FOXP3 phosphorylation at Ser422 mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.

Molecular mechanisms underlying the regulation and functional plasticity of FOXP3(+) regulatory T cells.

CD4+ CD25+ regulatory T (Treg) cells engage in the maintenance of immunological self-tolerance and homeostasis by limiting aberrant or excessive inflammation. The transcription factor forkhead box P3 (FOXP3) is critical for the development and function of Treg cells. The differentiation of the Treg cell lineage is not terminal, as developmental and functional plasticity occur through the sensing of inflammatory signals in the periphery. Here, we review the recent progress in our understanding of the molecular mechanisms underlying the regulation and functional plasticity of CD4+ CD25+ FOXP3+ Treg cells, through the perturbation of FOXP3 and its complex at a transcriptional, translational and post-translational level.

Inflammation negatively regulates FOXP3 and regulatory T-cell function via DBC1.

Significance Treg cells suppress excessive and aberrant immune responses. Impaired function or homeostasis of Treg cells would induce severe autoimmune and inflammatory diseases. Forkhead box P3 (FOXP3), as a master regulator of Treg cells, forms a large complex with other binding factors to modulate Treg-cell function subtly in pathological and physiological conditions. We identified that Deleted in breast cancer 1 (DBC1) is an essential subunit of the FOXP3 complex in human CD4+ Treg cells. Our results show that the inflammatory cytokines TNF-α or IL-6 trigger FOXP3 degradation, whereas downregulation of DBC1 expression prevents FOXP3 degradation and maintains Treg-cell function under inflammatory stimuli in vitro and in vivo. These findings unveil a previously unidentified pathway for therapeutically modulating FOXP3+ Treg-cell stability under inflammation. Forkhead box P3 (FOXP3)-positive Treg cells are crucial for maintaining immune homeostasis. FOXP3 cooperates with its binding partners to elicit Treg cells’ signature and function, but the molecular mechanisms underlying the modulation of the FOXP3 complex remain unclear. Here we report that Deleted in breast cancer 1 (DBC1) is a key subunit of the FOXP3 complex. We found that DBC1 interacts physically with FOXP3, and depletion of DBC1 attenuates FOXP3 degradation in inflammatory conditions. Treg cells from Dbc1-deficient mice were more resistant to inflammation-mediated abrogation of Foxp3 expression and function and delayed the onset and severity of experimental autoimmune encephalomyelitis and colitis in mice. These findings establish a previously unidentified mechanism regulating FOXP3 stability during inflammation and reveal a pathway for potential therapeutic modulation and intervention in inflammatory diseases.

60-kDa Tat-interactive Protein (TIP60) Positively Regulates Th-inducing POK (ThPOK)-mediated Repression of Eomesodermin in Human CD4+ T Cells

Background: ThPOK is required for CD4+ T helper cell differentiation and represses CD8 T cell-related genes, including Eomesodermin (Eomes) and IFNγ. Results: TIP60 stabilizes ThPOK by acetylation and augments ThPOK-mediated transcriptional repression of Eomes. Conclusion: TIP60 is a positive regulator of ThPOK. Significance: TIP60 could be a novel target for modulating IFNγ-mediated inflammation against cancer and virus infection. The abundant expression of IFNγ in Th-inducing POK (ThPOK)-deficient CD4+ T cells requires the activation of Eomesodermin (Eomes); however, the underlying mechanism of this phenomenon remains unclear. Here we report that ThPOK binds directly to the promoter region of the Eomes gene to repress its expression in CD4+ T cells. We identified the histone acetyltransferase TIP60 as a co-repressor of ThPOK-target genes, where ectopically expressed TIP60 increased ThPOK protein stability by promoting its acetylation at its Lys360 residue to then augment the transcriptional repression of Eomes. Moreover, knockdown of endogenous TIP60 abolished the stabilization of ThPOK in CD4+ T cells, which led to the transcriptional activation of Eomes and increased production of IFNγ. Our results reveal a novel pathway by which TIP60 and ThPOK synergistically suppresses Eomes function and IFNγ production, which could contribute to the regulation of inflammation.

USP22 is a positive regulator of NFATc2 on promoting IL2 expression

Nuclear factor of activated T cells (NFAT) is an important regulator of T cell activation. However, the molecular mechanism whereby NFATc2 regulates IL2 transcription is not fully understood. In this study, we showed that ubiquitin‐specific protease 22 (USP22), known as a cancer stem cell marker, specifically interacted with and deubiquitinated NFATc2. USP22 stabilized NFATc2 protein levels, which required its deubiquitinase activity. Consistent with these observations, depletion of USP22 in T cells reduced the expression of IL2, which is a cytokine that signifies T effector cell activation. Our findings thus unveil a previously uncharacterized positive regulator of NFATc2, suggesting that targeting the deubiquitinase activity of USP22 could have therapeutic benefit to control IL2 expression and T cell function.