An integrated transcription factor framework for Treg identity and diversity

Abstract

Vertebrate cell identity depends on the combined activity of scores of transcription factors (TF). While TFs have often been studied in isolation, a systematic perspective on their integration has been missing. Focusing on FoxP3+ regulatory T cells (Tregs), key guardians of immune tolerance, we combined single-cell chromatin accessibility, machine learning, and high-density genetic variation, to resolve a validated framework of diverse Treg chromatin programs, each shaped by multi-TF inputs. This framework identified previously unrecognized Treg controllers (Smarcc1) and illuminated the mechanism of action of FoxP3, which amplified a pre-existing Treg identity, diversely activating or repressing distinct programs, dependent on different regulatory partners. Treg subpopulations in the colon relied variably on FoxP3, Helios+ Tregs being completely dependent, but RORγ+ Tregs largely independent. These differences were rooted in intrinsic biases decoded by the integrated framework. Moving beyond master regulators, this work unravels how overlapping TF activities coalesce into Treg identity and diversity.

Keywords: autoimmunity; gene expression; immunoregulation.

Fig. 1.

Single-cell ATAC-seq reveals imbricated TF activities across diverse Treg cell states. (A) Experimental overview. Single-cell chromatin accessibility profiling was used to link TF activities to diverse Treg chromatin states. Continuous Treg cell states were first annotated using TF motif enrichment. Topic modeling was used to learn groups of covarying OCRs that formed discrete regulatory modules underlying observed cell states. Cis-regulatory variation in B6/Cast F1 hybrid scATAC-seq data enabled identification of causal regulators of Treg chromatin programs. The resulting Treg regulatory framework was validated using TF binding (ChIP-seq, CUT&RUN) and knockout datasets. All generated datasets and associated metrics are described in Dataset S1. (B) Aggregated accessibility profiles of splenic Treg and Tconv single cells at the Foxp3 locus from scATAC-seq data generated from a Foxp3^IRES-GFP reporter mouse; highlights indicate conserved noncoding sequence (CNS) loci previously described to control Foxp3 expression. (C) Relative accessibility (chromVAR scores) across Treg single cells of OCRs increased in accessibility in aTreg vs. rTreg populations (FoldChange > 2 in data from ref. 34) visualized on UMAP of splenic Treg scATAC-seq data. (D) Gene scores, chromatin-based proxies for gene expression, visualized for select genes on Treg UMAP from C. (E) Relative accessibility (chromVAR motif scores) of OCRs containing indicated TF motifs; motifs averaged within “archetypes” (35) to reduce redundancy. Only motifs whose corresponding TF(s) are expressed in Treg cells are shown. Motif logos are representative of TFs from each archetype.

Fig. 2.

Topic modeling and high-density genetic variation learn an integrated Treg TF framework. (A) Enrichment of TF motifs within distal OCRs from each topic; black indicates enrichment FDR < 1 × 10⁻¹⁰. Motifs are organized by TF family (row annotation). (B) Proportion of variance in accessibility explained by each topic in two independently generated spleen Treg scATAC datasets. (C) Topic-specific AME (FDR < 0.10) of motifs in each Treg topic. Heatmap in same order as in A and shows overlap between motif enrichment and significant AME scores. Positive AMEs (pink) indicate positive effect on chromatin accessibility and negative AMEs (green) indicate negative effect on chromatin accessibility. Motifs are ordered by TF family. (D) Gene Ontology gene sets significantly enriched among regulatory regions in each topic [using GREAT (61) analysis]. Heatmap indicates fold change of enrichment relative to background. Full table of enrichments and pathway names is provided in Dataset S5. (E) Enrichment [signed log₁₀(FDR), permutation test] in each topic of Gata3-dependent OCRs (GATA-motif containing OCRs decreased in accessibility > twofold in Foxp3-cre×Gata3^fl/fl Treg-specific Gata3 KO vs. Foxp3-cre×Gata3^+/+ WT scATAC-seq). Panel above indicates predicted topic effect based on topic TF framework in C. (F) Enrichment [signed log₁₀(FDR), permutation test] in each topic of c-Maf-dependent OCRs [MAF-motif containing OCRs decreased in accessibility > twofold in Foxp3-cre×Maf ^fl/fl Treg-specific c-Maf KO vs. Foxp3-cre×Maf ^+/+ WT (62)]. Panel above indicates predicted topic effect based on topic TF framework in C. (G) Enrichment [signed log₁₀(FDR), permutation test] in each topic of Smarcc1-dependent OCRs (OCRs with loss of accessibility at P < 0.05 in Foxp3^IRES-GFP Tregs electroporated with CRISPR/Cas9 ribonucleoprotein complexes carrying Smarcc1-targeting vs. control gRNAs and transferred for 1 wk into Treg-depleted Foxp3^DTR hosts). Panel above indicates predicted topic effect based on topic TF framework in C.

Fig. 3.

Adaptations to tissues and cytokines mapped to the Treg TF framework. (A) Proportion of variance in accessibility explained by each topic in colon or spleen Treg scATAC data (from the same Foxp3^IRES-GFP reporter mouse). (B) Differential accessibility per topic between aggregated colon and spleen Treg scATAC profiles. (C) Differential accessibility per motif in each topic (distal OCRs, FDR < 0.05) between colon and spleen Treg overlaid onto motif to topic connections from Fig. 2C (enrichment and F1 AME). Gray indicates significant topic-motif enrichment from Fig. 2A but no significant change in accessibility across colon vs. spleen comparisons. (D) Relative accessibility (chromVAR scores) of OCRs from Helios- (Topic 10) or RORγ- (Topic 9) specific topics across spleen and colon Treg single cells visualized on scATAC UMAP, separated by organ. Right panel indicates gene module scores for genes from a Helios vs. RORγ-specific gene expression signature visualized on the same UMAP.

Fig. 4.

FoxP3 effects and Treg identity in the Treg regulatory framework. (A) Experimental Scheme. FoxP3-deficient (KO) and -sufficient (WT) GFP+ Tregs were sorted along with GFP- Tconv from Foxp3^fs327-GFP/Foxp3-Thy1.1 or Foxp3^wt-GFP/Foxp3-Thy1.1 heterozygous female mice for multiplexed scATAC-seq. (B) Relative accessibility (chromVAR scores) across WT and KO Treg single cells of OCRs increased in accessibility in aTreg vs. rTreg populations, visualized on UMAP of scATAC of Treg and Tconv from FoxP3 WT or KO heterozygous female mice. (C) Proportion of rTreg and aTreg populations in FoxP3 WT or KO populations by CD44 and CD62L flow cytometry with quantification across biological replicates. (D) Differential accessibility per motif in each topic (distal OCRs, FDR < 0.05) between WT and KO Treg cells in rTreg or aTreg comparisons for motif to topic connections from Fig. 2C. Gray indicates significant motif to topic connection from Fig. 2C but no significant change in accessibility across FoxP3 comparisons. (E) Exemplar loci demonstrating FoxP3 positive (Left) and negative (Right) effects on accessibility and dependence on distinct cofactors as identified by the topic-motif Treg framework. (Top tracks) Aggregated scATAC-seq reads from WT Treg (red), KO Treg (blue), or Tconv (gray) cells from heterozygous female mice. (Middle) Treg ChIP-Seq or CUT&RUN tracks of indicated TFs identified as contributing to FoxP3 positive (red) or negative (blue) action. (Bottom) Treg H3K27Ac or FoxP3 HiChIP. RNA expression from aggregated scRNA-seq at these loci is displayed below. Highlights indicate OCRs belonging to Topics 3, 10, 14 (orange, left, positive FoxP3 effect) or Topics 16, 17 (blue, right, negative FoxP3 effect). (F) Differential accessibility per motif in each topic (distal OCRs, FDR < 0.05) between KO rTreg and Tconv cells for motif to topic connections from Fig. 2C. Gray indicates significant motif to topic connection from Fig. 2C but no significant change in accessibility across differential comparisons. (G) Normalized expression of TFs in scRNA-seq of early thymic Treg differentiation (86). TregP: Treg Progenitor; RT-Treg: recirculating/long-term resident Treg.

Fig. 5.

FoxP3-independent RORγ+ Treg-like cells. (A) Rorc gene scores and NR/19 (contains RORγ) motif accessibility (chromVAR score) in WT and KO Treg populations in scATAC-seq data from Fig. 4. (B) Proportion of RORγ+ vs. Helios+ Tregs among WT or KO Tregs in heterozygous female mice across organs. (C) Quantification of A. (D) Proportion of GFP+ Tregs among WT or KO Tregs with or without VNMA antibiotic treatment. (E) Proportion of RORγ+ vs. Helios+ Tregs among WT or KO colon Tregs before or after VNMA antibiotic treatment. (F) Proportion of IL17A+ RORγ+ cells among stimulated FoxP3 KO or WT Tregs from spleen or colon. (G) UMAP of scRNA-seq of colonic lamina propria Treg and Tconv from FoxP3 WT or KO heterozygous female mice, separated by genotype and cell type. Pink indicates Rorc+ reporter+ WT or KO Tregs, blue indicates all other reporter+ WT or KO Tregs, and orange indicates reporter- Tconv. (H) Il17a expression overlaid onto UMAP from G. (I) Distribution of Treg gene signature expression scores across indicated populations in colon scRNA-seq data from G. (J) Il10 expression overlaid onto UMAP from G. (K) Differential accessibility per motif in each topic (distal OCRs, FDR < 0.05) between WT RORγ+ and Helios+ aTreg cells for motif to topic connections from Fig. 2C. Gray indicates significant motif to topic connection from Fig. 2C but no significant change in accessibility across FoxP3 comparisons. (L) Differential accessibility per motif in each topic (aggregating motif family members contributing to each topic from Fig. 2C) in WT vs. Ikzf2 conditional KO (Top) or WT vs. Rorc conditional KO colon Treg scATAC data (39). Comparisons in outlined boxes are significant at FDR < 0.05.