Chromatin-modifying Complexes Research Articles

Interplay between transcriptional regulators and the SAGA chromatin modifying complex fine-tune iron homeostasis

The human fungal pathogen Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 (also known as Cap2), Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.

Epigenetic Dynamics in the Function of T-Lineage Regulatory Factor Bcl11b.

The transcription factor Bcl11b is critically required to support the development of diverse cell types, including T lymphocytes, type 2 innate lymphoid cells, neurons, craniofacial mesenchyme and keratinocytes. Although in T cell development its onset of expression is tightly linked to T-lymphoid lineage commitment, the Bcl11b protein in fact regulates substantially different sets of genes in different lymphocyte populations, playing strongly context-dependent roles. Somewhat unusually for lineage-defining transcription factors with site-specific DNA binding activity, much of the reported chromatin binding of Bcl11b appears to be indirect, or guided in large part by interactions with other transcription factors. We describe evidence suggesting that a further way in which Bcl11b exerts such distinct stage-dependent functions is by nucleating changes in regional suites of epigenetic modifications through recruitment of multiple families of chromatin-modifying enzyme complexes. Herein we explore what is - and what remains to be - understood of the roles of Bcl11b, its cofactors, and how it modifies the epigenetic state of the cell to enforce its diverse set of context-specific transcriptional and developmental programs.

Transcriptional Activation of MYC-Induced Genes by GCN5 Promotes B-cell Lymphomagenesis.

Overexpression of the MYC oncoprotein is an initiating step in the formation of several cancers. MYC frequently recruits chromatin-modifying complexes to DNA to amplify the expression of cancer-promoting genes, including those regulating cell cycle, proliferation, and metabolism, yet the roles of specific modifiers in different cancer types are not well defined. Here, we show that GCN5 is an essential coactivator of cell-cycle gene expression driven by MYC overexpression and that deletion of Gcn5 delays or abrogates tumorigenesis in the Eμ-Myc mouse model of B-cell lymphoma. Our results demonstrate that Gcn5 loss impacts both expression and downstream functions of Myc. SIGNIFICANCE: Our results provide important proof of principle for Gcn5 functions in formation and progression of Myc-driven cancers, suggesting that GCN5 may be a viable target for development of new cancer therapies.

Abstract PR05: Loss of PRC2 or KMT2D-COMPASS generates two quasi-mesenchymal cell states with distinct metastatic abilities

Abstract The epithelial-mesenchymal transition (EMT) is a key cell-biological program enabling carcinoma cell phenotypic plasticity. Accumulating evidence suggests EMT programs do not operate as a stereotypical program that functions as a binary switch, shifting cells from an epithelial (E) to a mesenchymal (M) state. Instead, EMT programs generate cells that enter into a series of intermediate states arrayed along the E-M phenotypic spectrum. At present, we still lack a coherent understanding of how carcinoma cells control their entrance into and residence in these various intermediate states, and which EMT intermediate state(s) favour the metastatic process. Here we characterize a new level of regulation of EMT, consisting of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, that function as critical regulators to maintain stable residence of both normal and neoplastic cells in an epithelial state via regulating the downstream response of EMT-inducing signals. Dysfunction of either of these two complexes causes cells that were previously stably ensconced in an epithelial state to lapse into two distinct quasi-mesenchymal cell states with strongly differing metastatic abilities. These observations uncover a novel mechanism that regulates E-M plasticity, specifies which intermediate EMT state a cell will reside in, and thereby determines the metastatic ability of carcinoma cells. Citation Format: Yun Zhang, Joana Liu Donaher, Sunny Das, Xin Li, Ferenc Reinhardt, Jordan A. Krall, Arthur W. Lambert, Prathapan Thiru, Heather R. Keys, Mehreen Khan, Matan Hofree, Molly M. Wilson, Ozlem Yedier-Bayram, Nathan A. Lack, Tamer T. Onder, Tugba Bagci-Onder, Michael Tyler, Itay Tirosh, Aviv Regev, Jacqueline Lees, Robert A. Weinberg. Loss of PRC2 or KMT2D-COMPASS generates two quasi-mesenchymal cell states with distinct metastatic abilities [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR05.

USP38 Couples Histone Ubiquitination and Methylation via KDM5B to Resolve Inflammation.

Chromatin modifications, such as histone acetylation, ubiquitination, and methylation, play fundamental roles in maintaining chromatin architecture and regulating gene transcription. Although their crosstalk in chromatin remodeling has been gradually uncovered, the functional relationship between histone ubiquitination and methylation in regulating immunity and inflammation remains unclear. Here, it is reported that USP38 is a novel histone deubiquitinase that works together with the histone H3K4 modifier KDM5B to orchestrate inflammatory responses. USP38 specifically removes the monoubiquitin on H2B at lysine 120, which functions as a prerequisite for the subsequent recruitment of demethylase KDM5B to the promoters of proinflammatory cytokines Il6 and Il23a during LPS stimulation. KDM5B in turn inhibits the binding of NF‐κB transcription factors to the Il6 and Il23a promoters by reducing H3K4 trimethylation. Furthermore, USP38 can bind to KDM5B and prevent it from proteasomal degradation, which further enhances the function of KDM5B in the regulation of inflammation‐related genes. Loss of Usp38 in mice markedly enhances susceptibility to endotoxin shock and acute colitis, and these mice display a more severe inflammatory phenotype compared to wild‐type mice. The studies identify USP38‐KDM5B as a distinct chromatin modification complex that restrains inflammatory responses through manipulating the crosstalk of histone ubiquitination and methylation.

The SAGA chromatin-modifying complex: the sum of its parts is greater than the whole.

There are many large protein complexes involved in transcription in a chromatin context. However, recent studies on the SAGA coactivator complex are generating new paradigms for how the components of these complexes function, both independently and in concert. This review highlights the initial discovery of the canonical SAGA complex 23 years ago, our evolving understanding of its modular structure and the relevance of its modular nature for its coactivator function in gene regulation.

Poly(ADP-ribose) Polymerase 1 (PARP1) restrains MyoD-dependent gene expression during muscle differentiation

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Function and regulation of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) deubiquitinase module

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) chromatin modifying complex is a critical regulator of gene expression and is highly conserved across species. Subunits of SAGA arrange into discrete modules with lysine aceyltransferase and deubiquitinase activities housed separately. Mutation of the SAGA deubiquitinase module can lead to substantial biological misfunction and diseases such as cancer, neurodegeneration, and blindness. Here, we review the structure and functions of the SAGA deubiquitinase module and regulatory mechanisms acting to control these.

The epigenomics of sarcoma.

Epigenetic regulation is critical to physiological control of development, cell fate, cell proliferation, genomic integrity and, fundamentally, transcriptional regulation. This epigenetic control occurs at multiple levels including through DNA methylation, histone modification, nucleosome remodelling and modulation of the 3D chromatin structure. Alterations in genes that encode chromatin regulators are common among mesenchymal neoplasms, a collection of more than 160 tumour types including over 60 malignant variants (sarcomas) that have unique and varied genetic, biological and clinical characteristics. Herein, we review those sarcomas in which chromatin pathway alterations drive disease biology. Specifically, we emphasize examples of dysregulation of each level of epigenetic control though mechanisms that include alterations in metabolic enzymes that regulate DNA methylation and histone post-translational modifications, mutations in histone genes, subunit loss or fusions in chromatin remodelling and modifying complexes, and disruption of higher-order chromatin structure. Epigenetic mechanisms of tumorigenesis have been implicated in mesenchymal tumours ranging from chondroblastoma and giant cell tumour of bone to chondrosarcoma, malignant peripheral nerve sheath tumour, synovial sarcoma, epithelioid sarcoma and Ewing sarcoma - all diseases that present in a younger patient population than most cancers. Finally, we review current and potential future approaches for the development of sarcoma therapies based on this emerging understanding of chromatin dysregulation.

Abstract A14: Next-generation sequencing of Bacille Calmette-Guerin (BCG)-unresponsive tumors identifies actionable alterations for potential targeted therapy in non-muscle invasive bladder cancer

Abstract Introduction and Objective: Intravesical BCG remains standard of care for patients with intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC). Although most patients initially respond to therapy, there is a group of patients who will experience recurrence and subsequent progression to invasive disease. There are limited treatment options for patients with BCG-unresponsive bladder cancer and alternative therapeutic strategies are needed. Next-generation sequencing helps identify and prioritize therapeutic opportunities for clinical trial of novel targeted agents. Methods: All patients with urothelial carcinoma of the bladder who progressed to muscle-invasive disease after at least one course of intravesical BCG were identified within our institutional genomic database and in The Cancer Genome Atlas (TCGA) dataset. DNA from BCG-treated, chemotherapy naive, secondary MIBC tumors and matched normal DNA underwent targeted exome capture sequencing or whole-exome sequencing. Potentially actionable genomic alterations were defined according to OncoKB levels of evidence for each alteration as a predictive biomarker (OncoKB.org). Results: The cohort comprised 77 secondary MIBC specimens, each from a unique patient. Sixty-six (86%) were male and the median age was 67 years (IQR 61-75). At least one potentially actionable genomic alteration was identified in 85% of the specimens. RTK/MAPK pathway alterations, most commonly FGFR3 (18%) and ERBB2 (15%), were seen in 42% of patients. PIK3CA mutations, seen in 21%, often co-occurred with RTK/MAPK alterations, potentially representing a resistance mechanism to FGFR or ERBB2-directed monotherapy. CDKN2A loss, hypothesized to be a driver of progression in FGFR3-mutated tumors, occurred in 10% of the samples. Alterations in cell cycle regulators were seen in 65% of specimens, of which 40% also had an intact RB gene and could potentially be vulnerable to CDK4/6 inhibitors. MDM2 amplification with “wild-type” TP53 was seen in 13% of specimens that might be sensitive to small-molecule antagonists. Inactivating mutations in histone acetyltransferases, CREBBP or EP300, occurred in 17% of specimens that might confer sensitivity to HDAC inhibitors. Truncating mutations in the SWI/SNF chromatin-modifying complex occurred in 33%, predominately in ARID1A, which we recently reported to be associated with worse recurrence-free survival after BCG. Truncating KDM6A mutations were seen in 19%, which along with ARID1A mutated tumors may be vulnerable to EZH2 inhibitors. Conclusion: Limited effective treatments beyond radical cystectomy are available for BCG-unresponsive NMIBC. Genomic profiling identified several actionable alterations that offer potential therapeutic strategies for management of BCG-unresponsive disease. Targeted therapy for NMIBC patients warrants further investigation as further advances are made in development of more selective systemic inhibitors and improved intravesical drug delivery. Citation Format: Aleksandra Walasek, Nima Almassi, Victor McPherson, Shawn Dason, Nicole Benfante, Tanya Klein, Michael F. Berger, Nikolaus Schultz, Guido Dalbagni, Dean F. Bajorin, Jonathan E. Rosenberg, Hikmat Al-Ahmadie, Bernard H. Bochner, David B. Solit, Gopakumar Iyer, Eugene J. Pietzak. Next-generation sequencing of Bacille Calmette-Guerin (BCG)-unresponsive tumors identifies actionable alterations for potential targeted therapy in non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A14.

Current understanding of plant Polycomb group proteins and the repressive histone H3 Lysine 27 trimethylation.

Polycomb group (PcG) proteins are highly conserved chromatin-modifying complexes that implement gene silencing in higher eukaryotes. Thousands of genes and multiple developmental processes are regulated by PcG proteins. As the first chromatin modifier been identified in model plant Arabidopsis thaliana, the methyltransferase CURLY LEAF (CLF) and its catalyzed histone H3 Lysine 27 trimethylation (H3K27me3) have already become well-established paradigm in plant epigenetic study. Like in animals, PcG proteins mediate plant development and repress homeotic gene expression by antagonizing with trithorax group proteins. Recent researches have advanced our understanding on plant PcG proteins, including the plant-specific components of these well-conserved protein complexes, the close association with transcription factors and noncoding RNA for the spatial and temporal specificity, the dynamic regulation of the repressive mark H3K27me3 and the PcG-mediated chromatin conformation alterations in gene expression. In this review, we will summarize the molecular mechanisms of PcG-implemented gene repression and the relationship between H3K27me3 and another repressive mark histone H2A Lysine 121 mono-ubiquitination (H2A121ub) will also be discussed.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single -helix that makes multiple contacts with -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

SummaryRegulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.

Research Highlights

Research Highlights

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The Chromatin Regulator ZMYM2 Restricts Human Pluripotent Stem Cell Growth and Is Essential for Teratoma Formation.

SummaryChromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.

Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

USP22 stabilizes FoxP3 to maintain Regulatory T cell function through a dual mechanism resulting in tumor immune evasion

Abstract Aggressive cancers are less sensitive to standard cancer treatments available, reducing overall survival rates and increasing relapse percentage in cancer patients. As a result, there is an increasing need for alternative therapeutics that will prolong the survival of patients. Within the last decade, methods to attenuate tumor immune evasion have become a centerpiece of tumor therapies. However, a major hurdle in tumor immunotherapy is the immunosuppression mediated by Regulatory T (Treg) cells, which can promote tumor progression through the suppression of tumor immune evasion. Therefore, the ability to modulate Treg function in the context of cancer could lead to more effective therapies. Here, we identify the ubiquitin-specific peptidase 22 (USP22), a member of the deubiquitination module of the SAGA chromatin modifying complex, as a specific regulator of Foxp3, the lineage-specifying transcription factor of Tregs. Treg-specific ablation of Usp22 in mice reduced Foxp3 at both the transcriptional and post-translational level, creating defects in their suppressive function and leading to spontaneous autoimmunity. Importantly, USP22 ablation protected against tumor growth in multiple cancer models. Collectively, our findings reveal a previously underappreciated physiological function of USP22 in maintaining Treg stability and identify USP22 as a potential target for cancer immunotherapy.

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Regulatory T cells (Tregs) are required to control immune responses and maintain homeostasis, but are a significant barrier to anti-tumor immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of pro-inflammatory properties2, can promote autoimmunity and/or facilitate more effective tumor immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. Despite improved functional genetic tools that now allow for systematic interrogation, dissection of the gene regulatory programs that modulate Foxp3 expression has not yet been reported. In this study, we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Tregs and applied this technology to perform a targeted loss-of-function screen of ~490 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We discovered several novel modulators including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin modifying complex, was discovered to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein and created defects in their suppressive function that led to spontaneous autoimmunity but protected against tumor growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Tregs could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Tregs. These results reveal novel modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.