Yayi Gao

FOXP3 and RORγt: Transcriptional regulation of Treg and Th17

FOXP3(+)CD4(+)CD25(+) Regulatory T (Treg) cells and IL-17 producing helper T cells (Th17) are critical subsets of T cells which play essential roles in immune homeostasis. The Forkhead family transcription factor FOXP3 is predominantly expressed in Treg cells, where the FOXP3 ensemble is essential for Treg cell development and function. As FOXP3 is to Treg cells, the orphan retinoic acid nuclear receptor (ROR) family transcription factor RORγt is essential for Th17 development and function. In this review, we summarize recent progress of our understanding towards the molecular mechanisms underlying the differentiation and function of FOXP3(+) Treg cells and RORγt expressing Th17 cells. These may provide new insights into therapeutic intervention and targeting of human immune-deficient diseases.

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.

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.

Negative Regulation of Interferon-induced Transmembrane Protein 3 by SET7-mediated Lysine Monomethylation

Background: IFITM3 is a general antiviral host restriction factor against RNA viruses. Results: SET7-mediated monomethylation of IFITM3 at Lys-88 negatively affected its antiviral activity toward vesicular stomatitis virus (VSV) and influenza A virus (IAV) infection. Conclusion: The monomethylation of antiviral host restriction factors may perturb their function. Significance: Targeting the SET7 pathway could provide new antiviral therapeutic strategies. Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases.

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.

Reciprocal regulation of RORγt acetylation and function by p300 and HDAC1

T helper 17 (Th17) cells not only play critical roles in protecting against bacterial and fungal infections but are also involved in the pathogenesis of autoimmune diseases. The retinoic acid-related orphan receptor (RORγt) is a key transcription factor involved in Th17 cell differentiation through direct transcriptional activation of interleukin 17(A) (IL-17). How RORγt itself is regulated remains unclear. Here, we report that p300, which has histone acetyltransferase (HAT) activity, interacts with and acetylates RORγt at its K81 residue. Knockdown of p300 downregulates RORγt protein and RORγt-mediated gene expression in Th17 cells. In addition, p300 can promote RORγt-mediated transcriptional activation. Interestingly, the histone deacetylase (HDAC) HDAC1 can also interact with RORγt and reduce its acetylation level. In summary, our data reveal previously unappreciated posttranslational regulation of RORγt, uncovering the underlying mechanism by which the histone acetyltransferase p300 and the histone deacetylase HDAC1 reciprocally regulate the RORγt-mediated transcriptional activation of IL-17.

The Deubiquitinase USP17 Regulates the Stability and Nuclear Function of IL-33

IL-33 is a new member of the IL-1 family cytokines, which is expressed by different types of immune cells and non-immune cells. IL-33 is constitutively expressed in the nucleus, where it can act as a transcriptional regulator. So far, no direct target for nuclear IL-33 has been identified, and the regulation of IL-33 nuclear function remains largely unclear. Here, we report that the transcription of type 2 inflammatory cytokine IL-13 is positively regulated by nuclear IL-33. IL-33 can directly bind to the conserved non-coding sequence (CNS) before the translation initiation site in the IL13 gene locus. Moreover, IL-33 nuclear function and stability are regulated by the enzyme ubiquitin-specific protease 17 (USP17) through deubiquitination of IL-33 both at the K48 and at the K63 sites. Our data suggest that IL13 gene transcription can be directly activated by nuclear IL-33, which is negatively regulated by the deubiquitinase USP17.

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.

DNMT1 cooperates with MBD4 to inhibit the expression of Glucocorticoid‐induced TNFR‐related protein in human T cells

Glucocorticoid‐induced TNFR‐related protein (GITR) is constitutively expressed in T regulatory (Treg) cells and regulates their suppressive function. We identified two methylated CpG islands in the Gitr locus. Using a ChIP assay, we demonstrate that both DNMT1 and methyl‐CpG‐binding domain Protein 4 (MBD4) bind to the Gitr promoter. Moreover, knockdown of DNMT1 decreases the binding activity of MBD4. We observed much higher levels of both DNMT1 and MBD4 in human CD4+CD25− conventional T (Tconv) cells. Moreover, co‐overexpression of DNMT1 and MBD4 in Treg cells significantly inhibits GITR expression and impairs their suppressive activity. Our results reveal a novel molecular mechanism by which MBD4 inhibits GITR expression in a DNMT1‐dependent manner.