Ultrastable and Versatile Multimeric Ensembles of FoxP3 on Microsatellites

. Author manuscript; available in PMC: 2026 Apr 2.

Published in final edited form as: Mol Cell. 2025 Apr 2;85(8):1509–1524.e7. doi: 10.1016/j.molcel.2025.03.005

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
PE anti-mouse CD4 Antibody	Biolegend	Cat# 100408
APC/Cyanine7 anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody	Biolegend	Cat# 202520
FoxP3 Monoclonal Antibody (FJK-16s), Alexa Fluor 700	eBioscience	Cat# 56–5773-82
Brilliant Violet 421^™ anti-mouse CD152 Antibody	Biolegend	Cat# 106311
Pacific Blue^™ anti-mouse CD25 Antibody	Biolegend	Cat# 102022
Ultra-LEAF^™ Purified anti-mouse CD3ε Antibody	Biolegend	Cat# 100340
Ultra-LEAF^™ Purified anti-mouse CD28 Antibody	Biolegend	Cat# 102116
eBioscience^™ Fixable Viability Dye eFluor^™ 506	Invitrogen	Cat# 65–0866-14
Chemicals, Peptides, and Recombinant Proteins
Ni-NTA agarose	QIAGEN	Cat# 30250
HRV 3C protease	Homemade	N/A
HiTrap Heparin	GE Healthcare	Cat# 17040601
Superdex 200 Increase 10/300	GE Healthcare	Cat# 28990944
Superdex 75 Increase 10/300	GE Healthcare	Cat# 29148721
HiTrap SP	GE Healthcare	Cat# 17505301
Recombinant Histone Octamer (H3.1)	Active motif	Cat# 31870
H3K27ac-modified histone octamer	Epicypher	N/A
Lipofectamine 2000	Invitrogen	Cat# 11668027
SYBR Gold stain	Invitrogen	Cat# S11494
Phusion polymerase	New England Biolabs	Cat# M0530L
XhoI	New England Biolabs	Cat# R1046
BamHI-HF	New England Biolabs	Cat# R3136
EcoRI-HF	New England Biolabs	Cat# R3101
KpnI-HF	New England Biolabs	Cat# R3142S
HindIII-HF	New England Biolabs	Cat# R3104S
Deoxyribonuclease	Sigma	Cat# DN25
Bis-Tris native PAGE, 3–12%	Invitrogen	Cat# BN1003BOX
Novex TBE Gels, 6%	Invitrogen	Cat# EC62655BOX
Mini-PROTEAN TGX Precast Protein Gels,4–15%	BioRad	Cat# 4561086
Carbon Support Film 400mesh copper grids	Electron Microscopy Sciences	CF400H-CU
C-flat 300 mesh copper grids (CF-1.2/1.3)	Electron Microscopy Sciences	CF413–50
Uranyl formate	Electron Microscopy Sciences	Cat# 22451
Glutaraldehyde solution	Sigma	G5882
TriDye^™ Ultra Low Range DNA Ladder	New England Biolabs	Cat# N0558S
Normal Mouse Serum	Thermo Fisher	Cat# 10410
Recombinant Human IL-2	Peprotech	Cat# 200–02
Neutravidin Protein	Thermo Fisher Scientific	Cat#31000
Catalase from bovine liver	Sigma-Aldrich	Cat#C3155
Glucose Oxidase from Aspergillus niger	Sigma-Aldrich	Cat#G2133
Bovine Serum Albumin	Millipore Sigma	Cat#10711454001
Quartz slides	G Finkenbeiner	Cat#100
Micro Cover Glasses	VWR	Cat#48393
N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane	EMD Millipore	Cat#8.19172.0100
Alconox Powder Detergent	Sigma-Aldrich	Cat#242985
Trolox ^®, 97%	Fisher scientific	Cat#AC218940050
Sulfo-Cyanine5 NHS ester	Lumiprobe	Cat#23320
Sulfo-Cyanine3 NHS ester	Lumiprobe	Cat#21320
Sulfo-Cyanine7 NHS ester	Lumiprobe	Cat#15320
DNA retardation gels (6%)	Thermo Scientific	Cat#EC63652BOX
Low Molecular Weight DNA ladder	New England Biolabs	Cat#N3233S
Potassium hydroxide	Sigma-Aldrich	Cat#221473
Epoxy	Devcon	Cat#14250
Biotin-PEG-SVA, MW 5,000	Laysan Bio Inc.	Cat#Biotin-PEG-SVA5000–100mg
mPEG-Succinimidyl Valerate, MW 5,000	Laysan Bio Inc.	Cat#MPEG-SVA-5000–1g
TetraSpeck^™ Microspheres, 0.1 μm, fluorescent blue/green/orange/dark red	Thermo Fisher Scientific	Cat#T7279
Critical Commercial Assays
QIAquick Nucleotide Removal kit	QIAGEN	Cat# 28304
Dual-Luciferase^® Reporter Assay System	Promega	Cat# E1910
Dynabeads^™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation	Thermofisher	Cat# 11452D
Naive CD4+ T Cell Isolation Kit, mouse	Miltenyi Biotec	Cat# 130–104-453
FoxP3 / Transcription Factor Staining Buffer Set	eBioscience^™	Cat# 00–5523-00
Deposited Data
FOXP3^ΔN_TTG₂₄ coordinates and map	This study	EMDB-46499; PDB: 9D2L
FOXP3^ΔN_TTTTG₁₅_3DNA coordinates and map	This study	EMDB-46480; PDB:9D22
FOXP3^ΔN_TTTTG₁₅_2DNA_1 coordinates and map	This study	EMDB-46493; PDB:9D2E
FOXP3^ΔN_TTTTG₁₅_2DNA_2 coordinates and map	This study	EMDB-46498; PDB: 9D2J
Experimental Models: Cell Lines
HEK293T cells	This study	N/A
CD4⁺ T cells	This study	N/A
Experimental Models: Organisms/Strains
E. coli BL21 (DE3)	Stratagene	Cat# 230130
C57BL/6 mice	This study	N/A
Oligonucleotides
DNAs	See Table S1	N/A
Cy5-DNAs	See Table S1	N/A
Biotin-Cy3-Cy5-DNAs	See Table S1	N/A
Biotin-Cy5-DNAs	See Table S1	N/A
Cy7-DNAs	See Table S1	N/A
Recombinant DNA
pET50b-FoxP3^ΔN	This study	N/A
pET50b-FoxP3^ΔN-F331D	This study	N/A
pET50b-FoxP3^ΔN-Y330A/F331A/Y333A	This study	N/A
pcDNA3.1+	This study	N/A
pcDNA-HA-FoxP3	This study	N/A
pcDNA-HA-FoxP3-F331D	This study	N/A
MSCV-IRES-Thy1.1	This study	N/A
MSCV-FoxP3-IRES-Thy1.1	This study	N/A
MSCV-FoxP3-F331D-IRES-Thy1.1	This study	N/A
MSCV-FoxP3-Y330A/F331A/Y333A-IRES-Thy1.1	This study	N/A
PGL4.31	This study	N/A
PGL4.31_enhancer_random	This study	N/A
PGL4.31_enhancer_(T3G)₁₂	This study	N/A
PGL4.31_promoter_(T3G)₁₂	This study	N/A
PGL4.31_enhancer_(T3G)₂₄	This study	N/A
PGL4.31_promoter_(T3G)₂₄	This study	N/A
PGL4.31_enhancer_601	This study	N/A
PGL4.31_promoter_601	This study	N/A
Software and Algorithms
CryoSparc	Punjani et al., 2017	https://cryosparc.com/
COOT	Emsley, et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PHENIX	Liebschner et al., 2019	https://www.phenix-online.org/
Pymol	Schrödinger, LLC.	https://pymol.org/2/
UCSF Chimera	Pettersen et al., 2004	https://www.cgl.ucsf.edu/chimera/
FlowJo	FlowJo LLC	https://www.bdbiosciences.com/en-ca/products/software/flowjo-v10-software
bowtie2 (v2.3.4.3)⁴⁸	Langmead and Salzberg, 2012	http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
HOMER (v4.10.3)⁴⁹	Heinz S et al., 2010	http://homer.ucsd.edu/homer/index.html
MEME (v5.3.3)⁵⁰	Bailey et al., 2015	https://meme-suite.org/meme/
FIMO (v5.3.3)	Grant et al., 2011	https://meme-suite.org/meme/doc/fimo.html
Bedtools (v2.30.0)⁵¹	Aaron R et al., 2010	https://bedtools.readthedocs.io/en/latest/
Trimmomatic (v0.36)	Bolger et al., 2014	http://www.usadellab.org/cms/?page=trimmomatic
MACS2 (v2.1.1.20160309)	Zhang et al., 2008	https://pypi.org/project/MACS2/
Analysis codes	This study	https://github.com/DylannnWX/Hurlab_Nucleosome_Foxp3_Manuscript; Deposited at Zenodo (https://doi.org/10.5281/zenodo.14968234)