Supplementary Figure 1. Efficiency of Mll4 deletion and its effect on T cell populations in the periphery. Nature Immunology: doi: /ni.

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1 Supplementary Figure 1 Efficiency of Mll4 deletion and its effect on T cell populations in the periphery.

2 Expression of Mll4 floxed alleles (16-19) in naive CD4 + T cells isolated from lymph nodes and spleens of Mll4 fl/fl Cd4-Cre, Mll4 fl/fl Cd4-Cre +, Mll4 fl/+ Cd4-Cre + and Mll4 +/+ Cd4-Cre + mice. n = 3 independent experiments. Error bars: standard deviations. Distribution of RNA-Seq read density across Mll4 for sorted naive CD4 + CD62L + T cells from lymph nodes and spleen of Mll4 fl/fl Cd4- Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) mice. Red rectangle: the deleted exons of the Mll4 gene. Western Blot for Mll4, Rbbp5 and actin beta protein amount in naïve CD4 + T cells of Mll4 +/+ Cd4-Cre + (WT) and Mll4 fl/fl Cd4-Cre+ (Mll4KO). n = 2 independent experiments. Two gating strategies for CD4 + T cells. L/D: dead cell stain. Representative flow cytometry plots of CD4 + and CD8 + T cell populations in lymph nodes isolated from Mll4 fl/fl Cd4-Cre, Mll4 fl/fl Cd4- Cre +, Mll4 fl/+ Cd4-Cre + and Mll4 +/+ Cd4-Cre + mice. Shown is one representative experiment (n=5 independent experiments). Representative flow cytometry plots of CD4 + Foxp3 + cells isolated from lymph nodes as in (d). Gated CD4 + T cells from (d). Shown is one representative experiment (n=5 independent experiments). Quantification of total CD4 +, CD8 + and CD4 + Foxp3 + cell numbers shown in (d) and (e). ****P (Kruskal-Wallis test), error bars: standard deviations. Center line: mean.

3 Supplementary Figure 2 Cytokine expression by CD4 + T cells and characterization of T reg cells in the periphery of Mll4-KO mice.

4 a) Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in the spleen of Mll4 fl/+ Cd4-Cre + and Mll4 fl/fl Cd4-Cre + mice. Shown is one representative experiment (n=3 independent experiments). b) Percentages of IFN-γ- and IL-17a-producing CD4 + T cells in the spleen of Mll4 fl/+ Cd4-Cre + and Mll4 fl/fl Cd4-Cre + mice. P value n.s. (Mann-Whitney test). Error bars: standard deviations. Center line: mean. c) Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in mesenteric lymph nodes of Mll4 fl/+ Cd4-Cre + and Mll4 fl/fl Cd4-Cre + mice. Shown is one representative experiment (n=3 independent experiments). d) Percentages of IFN-γ- and IL-17a-producing CD4 + T cells in mesenteric lymph nodes of Mll4 fl/+ Cd4-Cre + and Mll4 fl/fl Cd4-Cre + mice. P value n.s. (Mann-Whitney test). Error bars: standard deviations. Center line: mean. e) Flow cytometry analysis of Ki67 in lymph nodes (right column), spleen (middle column) and thymus (left column) of Mll4 fl/+ Cd4- Cre + and Mll4 fl/fl Cd4-Cre + mice. Shown is one representative experiment (n=3 independent experiments). f) Flow cytometry analysis of Helios in lymph nodes (right column), spleen (middle column) and thymus (left column) of Mll4 fl/+ Cd4- Cre + and Mll4 fl/fl Cd4-Cre + mice. Shown is one representative experiment (n=3 independent experiments). g) In vitro suppression assay of WT and Mll4KO Treg (CD4 + CD25 + ) cells isolated from lymph nodes and spleen of Mll4 fl/fl Cd4-Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) mice, incubated at indicated ratios in the presence of APC and anti-cd3 antibodies and assessed by CFSE dilutions at day 3 of culture. n = 3 independent experiments. Error bars: standard deviations.

5 Supplementary Figure 3 T reg cell specific deletion of Mll4 does not affect T reg cell function.

6 a) mrna levels of Mll4 exons in CD4 + Foxp3-Cre YFP (effector T cells, T eff) and CD4 + Foxp3-Cre YFP+ (T reg) cells sorted from lymph nodes and spleen of Mll4 +/+ Foxp3-Cre YFP+ (WT) and Mll4 fl/fl Foxp3-Cre YFP+ (Mll4KO) mice. Error bars: standard deviations. b) Representative flow cytometry plots of CD4 + Foxp3 + cells in the thymus of WT (n=2) and Mll4KO (n=2) mice. c) Percentages of CD4 + Foxp3 + in the thymus of WT (n=2) and Mll4KO (n=2) mice. P value n.s. (Mann-Whitney test). Error bars: standard deviations. d) Representative flow cytometry plots of CD4 + Foxp3 + cells in spleens of WT (n=2) and Mll4KO (n=2) mice. e) Percentages of CD4 + Foxp3 + in spleens of WT (n=2) and Mll4KO (n=2) mice. P value n.s. (Mann-Whitney test). Error bars: standard deviations. f) Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in the spleen of Mll4 +/+ Foxp3-Cre YFP+ (n=2) and Mll4 fl/fl Foxp3-Cre YFP+ (n=2) mice. g) Representative flow cytometry plots of CD4 + Foxp3 + cells in peripheral lymph nodes of WT (n=2) and Mll4KO (n=2) mice. h) Percentages of CD4 + Foxp3 + in peripheral lymph nodes of WT (n=2) and Mll4KO (n=2) mice. P value n.s. (Mann-Whitney test). Error bars: standard deviations. i) Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in peripheral lymph nodes of Mll4 +/+ Foxp3-Cre YFP+ (n=2) and Mll4 fl/fl Foxp3-Cre YFP+ (n=2) mice. j) In vitro suppression assay of WT and Mll4KO T reg (CD4 + Foxp3 YFP+ CD25 + ) cells isolated from lymph nodes and spleen of Mll4 +/+ Foxp3-Cre YFP+ (WT) and Mll4 fl/fl Foxp3-Cre YFP+ (Mll4KO) mice, incubated at indicated ratios in the presence of APC and anti-cd3 antibodies and assessed by CFSE dilutions at day 3 of culture. n = 3 independent experiments. Error bars: standard deviations.

7 Supplementary Figure 4 Effect of Mll4 deletion on in vitro CD4 + T cell cultures.

8 a) Flow cytometry analysis of intracellular staining for FoxP3 and CFSE during in vitro differentiation of Mll4 fl/fl Cd4-Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) naive CD4 + T cells in T reg inducing conditions, with Foxp3 expression and CFSE dilutions measured at indicated time points of differentiation. Representative of n=2 independent experiments. b) IFN-γ (left column) and TBET (right column) expression in Th1 cells generated in vitro for 3 days from Mll4f l/fl Cd4-Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) naive CD4 + T cells. n=3 independent experiments. Error bars: standard deviations. c) Representative flow cytometry plots of TBET and IFN-γ performed on TH1 cells generated like in (b). Shown is one representative experiment (n=2 independent experiments). d) IL-17a (left column) and RORC (right column) expression in TH17 cells generated in vitro for 3 days from Mll4 fl/fl Cd4-Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) naive CD4 + T cells. n=3 independent experiments. Error bars: standard deviations. e) Representative flow cytometry plots of CD4 and IL-17a performed on TH17 cells generated like in (d). Shown is one representative experiment (n=2 independent experiments). f) IL-4 (left column) and GATA3 (right column) expression in TH2 cells generated in vitro for 3 days from Mll4 fl/fl Cd4-Cre (WT) and Mll4 fl/fl Cd4-Cre + (Mll4KO) naive CD4 + T cells. n=3 independent experiments. Error bars: standard deviations. g) Representative flow cytometry histogram of GATA3 performed on TH2 cells generated like in (f). Shown is one representative experiment (n=3 independent experiments).

9 Supplementary Figure 5 MLL4 binds a small portion of non-promoter H3K4me1 regions in naïve CD4 + T cells.

10 Western blot for H3K4me1, H3K4me2, H3K4me3 and histone H3 in Naïve CD4 + T cells of WT (Mll4 fl/fl Cd4-Cre ) and Mll4KO (Mll4 fl/fl Cd4-Cre + ) mice. Representative of n = 4 independent experiments for H3K4me1 and n = 2 for H3K4me2 and H3K4me3 with similar results. Numbers below Western blot image: ratio of H3K4me to H3 intensity bands normalized to WT cells by ImageJ. Mll3 expression in naïve CD4 + T cells and in vitro generated TH1, TH2, TH17 and T reg cells for 3 days. n = 3 independent experiments. Error bars: standard deviations. Pie chart of genomic distribution of MLL4 binding sites under stringent settings in naïve CD4 + T cells. Comparison of the distributions of ChIP-Seq read density of MLL4 and input for MLL4 peaks called by using SICER (applied to the MLL4 ChIP-Seq data) under different E-values from 1,000, 10,000, 100,000 to 1,000,000. For E-value = 1,000, the distributions are for all predicted peaks, and for other E-values the distributions are for the additional peaks called with the current E-value over the previous lower E-value. Percentages of H3K4me1-enriched genomic regions not bound by MLL4, predicted from ChIP-Seq data with the least stringent settings, controlled by input control. GREAT gene ontology enrichment analysis on biologic processes for non-promoter H3K4me1 enriched regions not bound by MLL4. Shown are the top five enriched ontologies. Average of 30-way phastcon conservation score on genomic sequences for non-promoter H3K4me1-enriched regions not bound by MLL4. UCSC genome browser images showing the ChIP-Seq read density distribution of MLL4, H3K4me1, and DNase-Seq read density across a genomic region around Foxp3. Rectangles: H3K4me1 enriched regions that were not bound by MLL4. Representatives of n = 2 for MLL4 and H3K4me1 and n = 1 for DNase-Seq and H3K27ac.

11 Supplementary Figure 6 Mll4 deletion induces a reduction in H3K4me1 and chromatin interaction.

12 A UCSC genome browser image showing the distributions of ChIP-Seq reads for MLL4 and H3K4me1 in a genomic region containing the gene Dynlt1f in WT and Mll4KO naïve CD4 + T cells. Blue rectangle: promoter region of Dynlt1f with the presence of MLL4 binding and H3K4me1 signals in the WT cells and increased H3K4me1 in the Mll4KO cells; Solid green rectangles: putative enhancers upstream of Dynlt1f with the presence of MLL4 binding and H3K4me1 peaks in the WT cells and decreased H3K4me1 in the Mll4KO cells; Red rectangles: putative enhancer regions upstream of Dynlt1f with no or background level of MLL4 binding but with H3K4me1 peaks in the WT cells and decreased H3K4me1 peaks in the Mll4KO cells. Representatives of n = 2 independent experiments. WT: Mll4 fl/fl Cd4-Cre and Mll4KO: Mll4 fl/fl Cd4-Cre + (throughout the figure). Cumulative distribution of normalized ChIP-Seq read densities for H3K4me1-enriched regions at promoter (upper panel) and nonpromoter (lower panel) regions in WT and Mll4KO naïve CD4 + T cells. P-value < 2.2E-16 for both panels by Kolmogorov-Smirnov test. Volcano plot showing both fold change (FC, x-axis) and FDR (y-axis) that quantify the significance of change in H3K4me1 levels for each H3K4me1 peak upon Mll4 deletion in naïve CD4 + T cells. Black dots: MLL4-unbound H3K4me1 peaks; Red dots: MLL4-bound H3K4me1 peaks; %s: the fraction of H3K4me1 peaks with significant changes upon Mll4 deletion (FDR < 1E-6 & FC > 2). Accumulative distribution of the fold change of H3K4me1 read density of non-promoter H3K4me1 peaks, sorted based on the number of distant interacting MLL4 sites (from 0 to >=3) based on interactions predicted from Hi-C data. Group 0 : a subset of MLL4- unbound sites that showed no PETs linking to other regions. P-value by Kolmogorov-Smirnov test: 6.1E-8 for >=3 vs 0, 1.2E-4 for 2 vs 0 and 3.8E-6 for 1 vs 0. UCSC genome browser images showing the distribution of read densities across genomic regions harboring Thy1 (left panel) and Foxp3 (right panel) for MLL4 ChIP-Seq in the control cells, and Hi-C interaction intensity, H3K27ac ChIP-Seq, and H3K27me3 ChIP-Seq in the control cells and in the Mll4KO cells. Representative of n = 3 for track Hi-C (WT), n = 2 for MLL4 (KO) track and H3K27me3 tracks and n = 1 for H3K27ac tracks. Accumulative distribution of the fold-change of H3K27ac ChIP-Seq read density at 2-kb bins overlapping with H3K27ac peaks that were sorted based on the decrease, increase or no response in interaction intensity upon Mll4 deletion, confined to genomic bins enriched with H3K27ac in either the KO or the control cells. P-value less than 2.2e-16 by Kolmogorov-Smirnov test for Decrease vs No change. Similar to (b), but the analysis was made for H3K27me3 on 2-kb bins overlapping with H3K27me3 peaks. P-value less than 2.2e-16 by Kolmogorov-Smirnov test for Decrease vs No change.

13 Supplementary Figure 7 MLL4 promotes the expression of super-enhancer targets through enhancer promoter interaction. Accumulative distribution of the fold-change of interaction at promoters of super-enhancer targets and at promoters of other genes upon Mll4 deletion. P-value is less than 2.2e-16 by Kolmogorov-Smirnov test. WT: Mll4 fl/fl Cd4-Cre and Mll4KO: Mll4 fl/fl Cd4-Cre + (throughout the figure). UCSC genome browser images showing the distribution of read densities across genomic regions harboring Cxcr4, Rac2 and Stk4 for MLL4 ChIP-Seq and H3K27ac ChIP-Seq in the control cells, Hi-C interaction density and RNA-Seq in the control cells and in the Mll4KO cells. Representatives of n = 3 for track Hi-C (WT), n = 2 for track Hi-C (KO), track MLL4, tracks RNA-Seq and n = 1 for track H3K27ac. Accumulative distribution of the fold-change of expression of genes sorted into four equal size groups based on the fold change of interaction intensity at promoters by Mll4 deletion in Naïve CD4 + T cells. A line shifting to the left means a more decrease in expression. P-value is less than 2.2e-16 by Kolmogorov-Smirnov test for Lowest vs Highest. Accumulative distribution of the fold-change of expression of super-enhancer targets and of other genes upon Mll4 deletion. P-value is less than 2.2e-16 by Kolmogorov-Smirnov test.

14 Supplementary Figure 8 Deletion of the MLL4-binding 8.5 region has no effect on T reg cell function or activity.

15 Flow cytometry analysis of Helios in T reg cells from thymus of Crispr WT control and Crispr KO mice. One representative plot of n=4. Flow cytometry analysis of Helios in T reg cells from spleen of Crispr WT control and Crispr KO mice. One representative plot of n=4. Flow cytometry analysis of Helios in T reg cells from lymph nodes of Crispr WT control and Crispr KO mice. One representative plot of n=4. Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in the spleen of Crispr WT (n=3) and Crispr KO (n=3) mice. Percentages of IFN-γ- and IL-17a-producing CD4 + T cells in the spleen of Crispr WT (n=3) and Crispr KO (n=3) mice. Center line: mean. P value n.s. (Mann-Whitney test). Error bars: standard deviations. Center line: mean. Representative flow cytometry plots of IFN-γ + and IL-17a + CD4 + T cells in peripheral lymph nodes of Crispr WT control (n=3) and Crispr KO (n=3) mice. Percentages of IFN-γ- and IL-17a-producing CD4 + T cells in peripheral lymph nodes of Crispr WT control (n=3) and Crispr KO (n=3) mice. P value n.s. (Mann-Whitney test). Center line: mean. Error bars: standard deviations. In vitro suppression assay of WT and Crispr KO T reg (CD4 + CD25 + ) cells isolated from lymph nodes and spleen of Crispr KO and Crispr WT mice. Effector CD4+CD25- T cells (T eff) were sorted form Crispr WT mice expressing CD45.1 and stained with CFSE. Suppression assay was set up for 3 days then proliferation of CD cells was assessed by FACS. Experiment was done in triplicates. Error bars: standard deviations. Center line: mean.

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