Extended Data Fig. 5: Usp22 regulates Foxp3 through transcriptional mechanisms.

a, Representative flow cytometry analysis of the YFP⁺ T_reg population (gated on CD4⁺ cells) from the spleen and lymph nodes of Usp22^+/+Foxp3^YFP-cre WT or Usp22^fl/flFoxp3^YFP-cre KO mice. b, Statistical analysis of YFP MFI in CD4⁺YFP⁺ T_reg cells from the thymus (Thy), peripheral lymph nodes (pLN) and spleen (Spl) of Usp22^+/+Foxp3^YFP-cre WT or Usp22^fl/flFoxp3^YFP-cre KO mice. c, Statistical analysis of CD4⁺YFP⁺ T_reg frequencies in Usp22^+/+Foxp3^YFP-cre WT or Usp22^fl/flFoxp3^YFP-cre KO mice, corresponding to b. d, Volcano plot for RNA sequencing of Usp22 RNP KO T_reg cells vs Rnf20 RNP KO T_reg cells. The x axis shows LFC; the y axis shows −log₁₀ of the P value as calculated by DESeq2. Genes downregulated in the Usp22 RNP KO compared with Rnf20 RNP KO are shown in red and upregulated genes are shown in blue, defined by P < 5 × 10⁻³ and LFC > 0.8. Foxp3 (shown in green) trended down but did not reach significance. e, qPCR analysis of FOXP3 mRNA in human T_reg cells from 2 donors 8 days post-electroporation with Cas9 RNPs targeting NTC, FOXP3, USP22, RNF20 or both USP22 and RNF20; normalized to the expression of ACTB transcripts. Data are mean ± s.e.m. and are representative of at least two independent experiments. f, qPCR analysis of Foxp3 mRNA levels in mouse T_reg cells 4 and 8 days post-electroporation with Cas9 RNPs targeting NTC, Foxp3, Usp22, Rnf20 or both Usp22 and Rnf20; normalized to the expression of Actb transcripts. g, Western blot analysis of ubiquitinated histone 2A (H2AK119Ub; H2A-ub) and ubiquitinated histone 2B (H2BK120Ub; H2B-ub) from iT_reg cells from Usp22^+/+Foxp3^YFP-cre WT or Usp22^fl/flFoxp3^YFP-cre KO mice. GAPDH was used as a loading control. Source data can be found in Supplementary Fig. 1. h, Schematic of Foxp3 locus depicting PCR products used for ChIP–qPCR in i and j. i, ChIP–qPCR analysis of H2AK119Ub (H2A-ub), where primers amplified across the TSS and the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ± s.d. j, ChIP–qPCR data analysis for H2BK120Ub (H2B-ub) for PCR across the TSS and across the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ± s.d. k, Heat map of ChIP–seq read density for Foxp3, Usp22 and Rnf20 at sites bound by Foxp3 (using previously published Foxp3 ChIP data⁵¹), ranked by highest to lowest Foxp3-binding signal. The corresponding LFC for either H2BK120Ub or H2AK119Ub upon Usp22 or Rnf20 deletion at these sites are plotted on the right, with each biological replicate shown as an individual column. l, Average ChIP–seq read density of H2BK120Ub at T_reg superenhancers in control versus Usp22-deficient T_reg cells. m, Co-occurrence analysis showing the natural log of the ratio of the observed number of overlapping regions over the expected values for sites that either gain or lose H2BK120Ub in Usp22-deficient T_reg cells against publicly available histone modification data for H3K4me, H3K4me3 and H3K27ac as well as enhancer classes, as described in Methods. n, Analysis of reciprocal regulation of Foxp3 by deubiquitinase Usp22 and E3 ubiquitin ligase Rnf20. YFP MFI of T_reg cells sorted from Usp22^+/+Foxp3^YFP-cre WT or Usp22^fl/flFoxp3^YFP-cre KO mice and then electroporated with either NTC or Rnf20 RNP, corresponding with Fig. 2j, where Foxp3 MFI from the same experiment is shown. All data are presented as mean ± s.e.m., unless otherwise stated. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in Methods, ‘Statistics and reproducibility’. Source data for blots can be found in Supplementary Fig. 1.

Source Data