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To extract protein, cells were treated in cold RIPA buffer containing protease inhibitor. To denature proteins, lysates were added to 4× loading buffer and heated to 95 °C for 10 min. Total cell lysate (30–50 µg) were loaded onto SDS PAGE gels, and then transferred to PVDF membranes. Blots were incubated with primary antibodies overnight at 4 °C, followed by detection with secondary antibody. The antibodies used were: MECOM (Cell Signaling Technology, #C50E12, 1:500 dilution in PBST), beta-Actin (Cell Signaling Technology, #4967, 1:1000 dilution in PBST), KDR/VEGFR2 (Santa Cruz, #sc-6251, 1:500 dilution in PBST), GAPDH (Santa Cruz, #sc-32233, 1:1000 dilution in PBST), alpha Tubulin (Santa Cruz, #sc-5286, 1:1000 dilution in PBST), AP1(Signaling Technology, #9165, 1:1000 dilution in PBST), goat anti-rabbit HRP-conjugated antibody (Jackson Labs, #111-035-144, 1:5000 dilution in PBST).

CUT&RUN was performed based on the CUTANA CUT&RUN protocol v1.6 (Epicypher). Isolated nuclei from FT282 and KURAMOCHI were bound to Concalavin A beads and incubated overnight at 4°C with primary antibody for H3K27me3 (Cell Signaling, RRID:AB_2798370), H3K27ac (Diagenode, RRID:AB_2637079), MECOM (Cell Signaling, AB_2184098), PAX8 (Novus, RRID:AB_2283498), SOX17 (Abcam, RRID:AB_2801385), WT1 (Santa Cruz, RRID:AB_632611). Samples were washed after overnight incubation and treated with pAG-Mnase for 10 minutes at room temperature followed by activation by calcium chloride for 2 hours at 4°C. The reaction was quenched with STOP buffer and DNA was extracted with a DNA purification kit (Epicypher). Library preparation was conducted with the NEBnext Ultra II kit with the following protocol adjustments. The second incubation during the end prep was extended to 1 hour at 50 degrees and all SPRI select steps were conducted at 1.5x volume. Cycling parameters were set to parameters as instructed in the CUT&RUN protocol v1.6 (Epicypher). Processing and peak calls were made using the same pipeline as the CUT&TAG pipeline.

For Western blot analyses, cells were harvested and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche). Protein samples were loaded on SDS-PAGE gels, transferred onto nitrocellulose membranes, and probed with the following antibodies: GAPDH (Cell Signaling, 8884; 1:1000 dilution), HA (BioLegend, 901501; 1:1000 dilution), LgBit (R&D systems, MAB10026, 1:1000 dilution), MECOM (Cell Signaling, 2593; 1:1000 dilution), PAX8 (Cell Signaling, 59019; 1:1000 dilution), PRDM3 (GenScript, U0869CG110-1; 1:10,000 dilution), VINCULIN (Sigma, V9131; 1:400 dilution), and HRP‐anti‐rabbit and HRP-anti-mouse (Cell Signaling).
RNA isolation was performed using QIAshredder (Qiagen) and RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s recommendations. qRT-PCR was performed with QuantStudio 6 Flex (Applied Biosystems) using iTaq Universal Probes One-Step Kit (Bio-Rad) and the following Taqman probes: PAX8 (IDT, Hs.PT.58.1610472) and MECOM (IDT, Hs.PT.58.39825759). Gene expression levels were normalized to the HPRT housekeeping gene (Applied Biosystems). For ChIP-qPCR, qPCRs were performed using the Fast SYBR Green master mix reagent (Roche) with primers indicated in Supplementary Data 5.