Fig 2: p40 up-regulates Setd1β expression in IECs. (A, B, D, and E) YAMC cells were treated with p40 at 10 ng/mL for the indicated times. (C) Enteroids and colonoids were generated and cultured as described in Figure 1, in the presence or absence of p40 (100 ng/mL). (A and C) RNA was isolated from (A) YAMC cells and (C) enteroids and colonoids for RT-PCR analysis of the levels of Setd1b mRNA. Setd1b mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. Data are quantified from 3 independent experiments. (C) For each experiment, data represent the average of at least 5 enteroids or colonoids in each group. (B, D, and E) Total cellular proteins were prepared from YAMC cells for Western blot analysis. β-actin blot was used as the protein loading control. The fold changes of band density are shown under the blot. Data are representative of at least 3 independent experiments. ∗P < .05 compared with the control group.

Fig 3: p40 supplementation in early life stimulates short-term up-regulation of Setd1b gene expression, but sustained increase in epigenetic marks in mice. The treatment plans for detecting the short-term (Figure 3A) and long-term effects (Figure 3B) are described. (A and B) RNA was isolated from colonic tissues for RT-PCR analysis of Setd1b gene expression. Each symbol represents one mouse in A and B. (C) Paraffin-embedded tissue sections were prepared for immunohistochemistry using anti-H3K4me1 antibody and fluorescein isothiocyanate–conjugated secondary antibody (green), an epithelial cell marker using anti–E-cadherin antibody and Cy3-conjugated secondary antibody (red), and nuclei using 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Images were taken using a fluorescent microscope at 40×. (D) The numbers of H3K4me1 and E-cadherin double-positive cells per crypt are shown. N = 5 in each group.

Fig 4: Transcription factor binding bypasses the requirement for KMT2A/B for bivalent gene activation.(a & b) Cell surface MHC-I in K-562 Cas9, KMT2B KO and KMT2A/KMT2B KO cells treated with 1µM VTP50469 (left), 3µM EPZ-011989 (middle) and combination (right), and (b) treated with 25ng/mL IFN-? for 48h. (c) Immunoblot of K-562 Cas9 and KMT2A/B KO cells treated with DMSO, 1µM VTP50469 or 3µM EPZ-011989. (d) Cell surface MHC-I in K-562 Cas9 and KMT2A/KMT2B KO cells transduced with lentiviral vectors encoding H3.3 WT or K27M and treated with 25ng/mL IFN-? for 48h. Representative plots from 2 experiments (Supplementary Figure 2). (e) Overcoming dependence on KMT2A/B is reliant on the NLCRC5-RFX5-enhanceosome. Cell surface MHC-I in K-562 Cas9 and KMT2A/KMT2B KO cells transduced with RFX5 sgRNA and treated with 3µM EPZ-011989 and 25ng/mL IFN-? (48h). (f & g) Genomic snapshots of MHC-I genes showing H3K4me3 CUT&Tag in K-562 Cas9 and KMT2A/B KO cells pre-treated with EPZ-011989 ± 25ng/mL IFN-? (48h). The EZH2i treated (no IFN-?) tracks are also shown in Fig. 7c. (h) Cell surface MHC-I in K-562 Cas9 and KMT2A/B KO cells transduced with control or SETD1A + SETD1B sgRNA, treated with EPZ-011989 and 25ng/mL IFN-? (48h). Representative plot from 4 experiments (Supplementary Figure 2). (a/b) and (e) are representative plots from 3 experiments (Supplementary Figure 2).

Fig 5: KMT2A/B is dispensable for MHC enhanceosome driven activation.(a) Schematic overview of cis-regulatory elements in the MHC-I promoter. NLRC5 forms an enhanceosome with the RFX (regulatory factor X) complex, made up of RFX5, RFXANK and RFAXP (RFX-associated ankyrin-containing protein); CREB (cAMP-responsive-element-binding); and NFY (nuclear transcription factor Y), which bind the SXY-molecule to activate transcription of MHC-I. (b) Immunoblot of K-562 Cas9 cells transduced with control and RFX5 sgRNA. (c) IFN-? time-course in K-562 Cas9 and indicated KO cells treated with 3µM EPZ-011989 and 25ng/mL IFN-? for the indicated time points. (d) Immunoblot of K-562 Cas9 and KMT2A/B KO cells transduced with control, SETD1A and/or SETD1B sgRNA.