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[Preprint]. 2025 Jan 14:2024.08.30.610436.

doi: 10.1101/2024.08.30.610436.

Cis-Regulatory Element and Transcription Factor Circuitry Required for Cell-Type Specific Expression of FOXP3

Jennifer M Umhoefer^{1

2

3}, Maya M Arce^{1

2

3}, Sean Whalen⁴, Rama Dajani¹, Laine Goudy^{1

2

3}, Sivakanthan Kasinathan^{1

5}, Julia A Belk⁶, Wenxi Zhang^{7

8}, Royce Zhou^{1

7}, Sanjana Subramanya¹, Rosmely Hernandez¹, Carinna Tran¹, Nikhita Kirthivasan^{1

3}, Jacob W Freimer^{1

2

9}, Cody T Mowery^{1

2

3}, Vinh Nguyen^{1

2

10

11

12}, Mineto Ota^{1

2

9}, Benjamin G Gowen^{13

14}, Dimitre R Simeonov^{2

3}, Gemma L Curie^{13

14}, Zhongmei Li^{1

2}, Jacob E Corn¹⁵, Howard Y Chang^{6

16}, Luke A Gilbert^{17

18}, Ansuman T Satpathy^{1

7

19}, Katherine S Pollard^{4

20

21

22}, Alexander Marson^{1

2

13

19

20

23

24}

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
² Department of Medicine, University of California, San Francisco, CA, USA.
³ Biomedical Sciences Graduate Program, University of California, San Francisco, CA, USA.
⁴ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
⁵ Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
⁶ Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA.
⁷ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
⁸ Tetrad Graduate Program, University of California, San Francisco, CA, USA.
⁹ Department of Genetics, Stanford University, Stanford, CA, USA.
¹⁰ Diabetes Center, University of California, San Francisco, CA, USA.
¹¹ UCSF CoLabs, University of California, San Francisco, CA, USA.
¹² Department of Surgery, University of California, San Francisco, CA, USA.
¹³ Innovative Genomics Institute, University of California, Berkeley, CA, USA.
¹⁴ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
¹⁵ Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, Switzerland.
¹⁶ Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
¹⁷ Arc Institute, Palo Alto, CA, USA.
¹⁸ Department of Urology, University of California, San Francisco, CA, USA.
¹⁹ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
²⁰ Institute for Human Genetics, University of California, San Francisco, CA, USA.
²¹ Department of Epidemiology and Biostatistics, University of California, San Francisco, CA, USA.
²² Chan Zuckerberg Biohub SF, San Francisco, CA, USA.
²³ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
²⁴ Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.

PMID: 39282425
PMCID: PMC11398386
DOI: 10.1101/2024.08.30.610436

Cis-Regulatory Element and Transcription Factor Circuitry Required for Cell-Type Specific Expression of FOXP3

Jennifer M Umhoefer et al. bioRxiv. 2025.

[Preprint]. 2025 Jan 14:2024.08.30.610436.

doi: 10.1101/2024.08.30.610436.

Authors

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
² Department of Medicine, University of California, San Francisco, CA, USA.
³ Biomedical Sciences Graduate Program, University of California, San Francisco, CA, USA.
⁴ Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
⁵ Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
⁶ Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA.
⁷ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
⁸ Tetrad Graduate Program, University of California, San Francisco, CA, USA.
⁹ Department of Genetics, Stanford University, Stanford, CA, USA.
¹⁰ Diabetes Center, University of California, San Francisco, CA, USA.
¹¹ UCSF CoLabs, University of California, San Francisco, CA, USA.
¹² Department of Surgery, University of California, San Francisco, CA, USA.
¹³ Innovative Genomics Institute, University of California, Berkeley, CA, USA.
¹⁴ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
¹⁵ Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, Switzerland.
¹⁶ Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
¹⁷ Arc Institute, Palo Alto, CA, USA.
¹⁸ Department of Urology, University of California, San Francisco, CA, USA.
¹⁹ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
²⁰ Institute for Human Genetics, University of California, San Francisco, CA, USA.
²¹ Department of Epidemiology and Biostatistics, University of California, San Francisco, CA, USA.
²² Chan Zuckerberg Biohub SF, San Francisco, CA, USA.
²³ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
²⁴ Department of Microbiology and Immunology, University of California, San Francisco, CA, USA.

PMID: 39282425
PMCID: PMC11398386
DOI: 10.1101/2024.08.30.610436

Abstract

FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Tregs). While mice exclusively express FOXP3 in Tregs, humans also transiently express FOXP3 in stimulated conventional CD4+ T cells (Tconvs). Mechanisms governing these distinct expression patterns remain unknown. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Tregs and Tconvs to discover cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv FOXP3 expression depended on a subset of Treg CREs and Tconv-selective positive (TcNS+) and negative (TcNS-) CREs. The CREs are occupied and regulated by TFs we identified as critical regulators of FOXP3. Finally, mutagenesis of murine TcNS- revealed that it is critical for restriction of FOXP3 expression to Tregs. We discover CRE and TF circuitry controlling FOXP3 expression and reveal evolution of mechanisms regulating a gene indispensable to immune homeostasis.

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Conflict of interest statement

A.M. is a cofounder of Site Tx, Arsenal Biosciences, Spotlight Therapeutics and Survey Genomics, serves on the boards of directors at Site Tx, Spotlight Therapeutics and Survey Genomics, is a member of the scientific advisory boards of Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen, and Tenaya, owns stock in Arsenal Biosciences, Site Tx, Cellanome, Spotlight Therapeutics, NewLimit, Survey Genomics, Tenaya and Lightcast and has received fees from Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, NewLimit, Abbvie, Gilead, Pfizer, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, Trizell, Vertex, Merck, Amgen, Genentech, GLG, ClearView Healthcare, AlphaSights, Rupert Case Management, Bernstein and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from the Parker Institute for Cancer Immunotherapy, the Emerson Collective, Arc Institute, Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem and reagents from Genscript and Illumina. C.T.M. is a Bio+Health Venture Fellow at Andreessen Horowitz. J.E.C. is a co-founder and SAB member of Serac Biosciences and an SAB member of Mission Therapeutics, Relation Therapeutics, Hornet Bio, and Kano Therapeutics. The lab of J.E.C. has funded collaborations with Allogene, Cimeio, and Serac. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, Cartography Biosciences, and Orbital Therapeutics, and is an advisor of 10x Genomics and Exai Bio. H.Y.C. was an advisor of Arsenal Bio and Chroma Medicine up to Dec. 15, 2024. H.Y.C. is an employee and stockholder of Amgen as of Dec. 16, 2024. J.W.F. was a consultant for NewLimit, is an employee of Genentech, and has equity in Roche. A.T.S. is a founder of Immunai, Cartography Biosciences, Santa Ana Bio, and Prox Biosciences, an advisor to 10x Genomics and Wing Venture Capital, and receives research funding from Astellas. J.M.U., A.M., M.A., L.G, and L.A.G. have filed patents related to this work.

Figures

**Figure 1.. CRISPRi tiling screen identifies FOXP3 CREs in Tregs and Tconvs.**
(A) Schematic depicting CRISPRi-based screens for FOXP3 CREs. (B) Top, full assayed region in CRISPRi *FOXP3* locus-tiling screen. Bottom subplot, -log10(p-value) of gRNA enrichment in FOXP3 high vs. low FACS bins in Treg and Tconv. Blue, gRNA enriched in FOXP3 High FACS bin. Red, gRNA enriched in FOXP3 Low FACS bin. Outlined, adjusted p-value ≤ 0.05. Treg CNS0 and CNS2, *FOXP3* TSS, the FLICR region, and TcNS+ are highlighted in grey. Treg, n = 2 donors; Tconv, n = 2 donors. Conservation track depicts vertebrate PhyloP 100-way conservation. (C and D) Arrayed validation of *FOXP3* TSS, FLICR, CNS0, and TcNS+ region-associated CRISPRi-responsive elements with paired Cas9 RNPs plotted along the *FOXP3* locus at 0 hours (Rest) and 48 hours (Stim) post-stimulation in Tregs (C) and Tconv (D). Locations of tiled deletions are indicated by line segments in (C) and (D). Lines indicate log2 fold change in mean FOXP3 MFI in Tregs (C) or %FOXP3+ Tconvs (D) in deletions vs. donor-specific paired *AAVS1* gRNA controls. Points indicate individual replicates of tiled deletions (Treg n = 2 donors; Tconv n= 3 donors). Positive CREs for each cell type are highlighted in red, negative CREs in blue, and non-regulatory regions in gray. (E) Flow plots of FOXP3 expression upon deletion of Tile A and an *AAVS1* control in resting Tregs. Tile letter corresponds to Tiled Deletions in (C, D). (F) Representative flow plots depicting FOXP3 expression with Tile C and paired *AAVS1* deletions in resting Tconv. Numbers indicate %FOXP3+ cells. Tile letter corresponds to Tiled Deletions in (C, D).

**Figure 2.. Positive and negative FOXP3 CREs map to active chromatin.**
(A) ATAC-seq in unstimulated (dark gray) and stimulated (light gray) Treg and Tconv. Positive CREs for the indicated cell type are highlighted in red, negative CRE in blue, and non-regulatory regions in gray. (B and C) ChIP-seq for H3K27ac in unstimulated (top) and stimulated (bottom) Tregs (B) and Tconvs (C). ChIP-seq is auto-scaled to the locus shown. (D) Post-bisulfite-conversion adapter tagging sequencing (PBAT-seq) percent methylation of CpGs at the *FOXP3* locus in naïve and activated Tregs (green) and naïve and activated Tconvs (gold) from a representative donor. PBAT-seq data is plotted as the mean percent methylation of 1500 bp bins with 300 bp step sizes across the locus shown.

**Figure 3.. CRISPRn TF screens identify trans-regulators of FOXP3 in Tconv.**
(A) Schematic depicting CRISPRn-based screens for FOXP3 trans-regulators in Tregs and Tconvs. (B and C) Volcano plots of log2 fold change of gene gRNA enrichment in FOXP3 high vs. low FACS bins versus -log10 of p-value in Tregs (B) and Tconvs (C). Color of points indicates significance (FDR ≤ 0.05). Blue, significantly enriched in FOXP3 high FACS bin. Red, significantly enriched in FOXP3 low FACS bin. Gray, not significant (Treg, n = 2 donors; Tconv n = 3 donors). (D) Top, log2 fold change of individual gRNA enrichment in FOXP3 high vs. low FACS bins for the top 12 significant FOXP3 positive and negative regulators in Tregs (left) and Tconvs (right, FDR ≤ 0.05; n = 2 Treg donors, n = 3 Tconv donors). gRNAs corresponding to the labelled significant gene are colored blue (gene enriched in FOXP3 high bin), red (gene enriched in FOXP3 low bin), or gray (NTC, non-targeting control gRNA). Bottom, distribution of gRNA in screen. (E and F) Arrayed validation of top FOXP3 positive and negative regulators at 48 hours poststimulation in Tregs (E) and Tconvs (F). Color indicates the directional effect in the screen (n = 2 donors x 2 gRNAs per target or 6 gRNAs targeting *AAVS1* control). (G) Selected flow plots of FOXP3, CBFB, *AAVS1*, SRF, and YBX1 KO in stimulated Tregs. (H) Selected flow plots of *AAVS1*, GATA3, ETS1, and MBD2 KO in Tconvs at 48 hours poststimulation. Numbers indicate %FOXP3+ cells.

**Figure 4.. KO of positive and negative trans-regulators change accessibility at CNS0 and TcNS+.**
(A) ChIP-seq in Tregs targeting Treg positive (red) and negative (blue) trans-regulators of FOXP3. CREs are colored based on the effect of the element. Red, positive regulator; blue, negative regulator; gray, not required for regulation. ChIP-seq is auto-scaled to the locus shown. (B and C) Publicly available ChIP-seq datasets in indicated T cells or T cell-based cell lines (B) or hematopoietic cell lines (C) targeting Tconv positive (red) and negative (blue) trans-regulators of FOXP3. CREs are labelled and scaling conducted as described in (A). (D) Effect of trans-regulator KO on DNA accessibility at the *FOXP3* locus. Bars beneath plots indicate significantly differentially accessible peaks in KOs vs. *AAVS1* controls (White, *Padj* ≤ 0.1; Black, *Padj* ≤ 0.05). For each KO comparison, n = 2–3 donors x 1 KO gRNA and 7–8 *AAVS1*-targeting gRNAs. Red, accessibility with indicated positive regulator KO; gray, accessibility in *AAVS1* controls. In CRISPRn TF KO screens, IL2RA and JAK3 were just above an FDR cutoff of 0.05.

**Figure 5.. TcNS- suppresses FOXP3 expression in murine Tconv.**
(A) Top, schematic showing location of GATA3 and STAT5 ChIP-seq in human Tconvs mapped to homologous regions in the murine genome. Below, ChIP-seq for H3K27ac (purple), GATA3, STAT5, and EGR2 in murine Tregs and CD4+ T cells. ChIP-seq is auto-scaled to the locus shown. (B) Allele effect size of TF binding motifs between human (reference allele) and mouse (alternative allele) TcNS-, plotted along the human locus. Allele effect size indicates the difference in motif scoring between the reference and alternate allele (alternate – reference). Only motifs corresponding to human FOXP3 trans-regulators are shown. (C) *Top*, percent FOXP3+ Tconvs with TcNS- tiled deletions. Dashed line indicates the mean %FOXP3+ cells in *Rosa26* controls, shown on the right. Locations of tiled deletions are indicated by line segments, which represent mean %FOXP3+ Tconvs. Points indicate individual replicates of tiled deletions (n = 2 replicates). Bottom, EGR2 ChIP-seq in murine CD4+ T cells. ChIP-seq is auto-scaled to the locus shown. (D) Flow plots depicting %FOXP3+ Tconvs with deletion of a segment in *Rosa26* or a tiled deletion at TcNS-.

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References

1. Farh K.K.-H., Marson A., Zhu J., Kleinewietfeld M., Housley W.J., Beik S., Shoresh N., Whitton H., Ryan R.J.H., Shishkin A.A., et al. (2015). Genetic and epigenetic fine mapping of causal autoimmune disease variants. Nature 518, 337–343. 10.1038/nature13835. - DOI - PMC - PubMed
1. Bulger M., and Groudine M. (2011). Functional and mechanistic diversity of distal transcription enhancers. Cell 144, 327–339. 10.1016/j.cell.2011.01.024. - DOI - PMC - PubMed
1. Hnisz D., Abraham B.J., Lee T.I., Lau A., Saint-André V., Sigova A.A., Hoke H.A., and Young R.A. (2013). Super-enhancers in the control of cell identity and disease. Cell 155, 934–947. 10.1016/j.cell.2013.09.053. - DOI - PMC - PubMed
1. Parker S.C.J., Stitzel M.L., Taylor D.L., Orozco J.M., Erdos M.R., Akiyama J.A., Lammerts Van Bueren K., Chines P.S., Narisu N., Program N.C.S., et al. (2013). Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. PNAS 110, 17921–17926. 10.1073/pnas.1317023110. - DOI - PMC - PubMed
1. Canver M.C., Smith E.C., Sher F., Pinello L., Sanjana N.E., Shalem O., Chen D.D., Schupp P.G., Vinjamur D.S., Garcia S.P., et al. (2015). BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197. 10.1038/nature15521. - DOI - PMC - PubMed

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This is a preprint.

Cis-Regulatory Element and Transcription Factor Circuitry Required for Cell-Type Specific Expression of FOXP3

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Cis-Regulatory Element and Transcription Factor Circuitry Required for Cell-Type Specific Expression of FOXP3

Authors

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Abstract

Conflict of interest statement

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