Transcriptional repressor, hypermethylated in cancer 1 (HIC1) is an important contributor to regulatory T (Treg) cell development and function. To investigate the mechanism by which it regulates Treg cell development, we systematically characterized the HIC1 interactome by affinity-purification mass spectrometry in human regulatory T cells. Interactors, involved in protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism processes were identified. These data demonstrate that HIC1 is a part of a FOXP3-RUNX1-CBFβ protein complex that regulates Treg signature genes and is indispensable for the suppressive function of FOXP3+ regulatory T cells.

The immunoprecipitated proteins were digested with trypsin. Briefly, urea buffer (8 M urea, 50mM Tris-HCl pH 8.0) was added to denature the proteins, followed by dithiothreitol (10 mM, 370C for 1 h), then iodoacetamide (~14 mM, RT 30 min in darkness). The samples were diluted to a urea concentration of less than 1 molar and then digested with sequencing grade modified trypsin (0.29 µg per sample) at 37 0C overnight (16-18 hours). The tryptic digests were acidified and desalted using in house made C18 Stage Tips (3M, Cat No 2215). LC-MS/MS analyses were conducted using Easy-nLC 1000 liquid chromatography (Thermo Scientific) coupled to a TSQ Vantage Triple Quadrupole mass spectrometer (Thermo Scientific). The column configuration included a 20 x 0.1 mm i.d. pre-column in conjunction with a 150 mm x 75 µm i.d. analytical column, both packed with 5 µm Reprosil C18-bonded silica (Dr Maisch GmbH). A separation gradient was used from 5% to 21% B in 11 min, then to 36% B in 9 min, to 100% in 2 min, then ending with an 8 min isocratic period. A flow rate of 300 nl/min was used. Selected reaction monitoring mass spectrometry was used to validate the relative abundance of BCOR, FOXP3, RUNX1, CBFΒ, IKZF3 and HIC-1 in immuno-precipitates from iTreg cells. ACTB and GAPDH were also measured as a reference protein. Heavy-labeled synthetic peptides (lysine 13C6 15N2 and arginine 13C6 15N4) were obtained for the targets of interest (PEPotec, Grade 2, Thermo Fischer Scientific). For these validations, IP’s were prepared from the differentiated iTreg cells from three donors.

CD4+ T cells were isolated from human umbilical cord blood, activated with plate-bound anti-CD3 (500 ng/24-well culture plate well; Immunotech) and soluble anti-CD28 (500 ng/mL; Immunotech) at a density of 2 × 10E6 cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum (10%). The media was cultured at 37°C in 5% CO2 and activated in presence of the following cytokines: IL-2 (12 ng/ml; R&D Systems); all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich); TGF-β1 (10 ng/ml; R&D Systems). HIC-1 immunoprecipitation (IP) was performed with an anti-HIC-1 antibody (Cat# sc-271499; Santa Cruz Biotechnology) that recognizes the C-terminus region of human HIC-1. The respective Immunoglobulin G (IgG) was used for the control IPs. Following the Pierce™ MS-Compatible Magnetic IP Kit (Cat# 90409) protocol, cells were harvested on ice and washed with PBS (2X). Cells were then re-suspended in the ice-cold IP-MS Cell Lysis Buffer, followed by incubation on ice for 20 min with periodic mixing. The protein lysate was centrifuged at 13,000 g for 10 min to pellet down the cell debris. The supernatant was transferred to a new microcentrifuge tube and protein concentration was determined by using a DC protein assay kit (BioRad). Cell lysates (500-1000 μg) were mixed with the IP antibody (1:50 dilution) and the antibody/lysate solution diluted with the IP-MS cell lysis buffer and incubated overnight at +4 °C to form the immune complex. The immune complex was then incubated with Protein A/G magnetic beads for 1 h at room temperature (RT), followed by washing of beads and elution. The eluate, containing the target antigen, was then transferred to a new low protein-binding microcentrifuge tube and dried with a vacuum concentrator prior to MS preparation.