Fig 2: RUNX1 positively transcriptional regulates the expression of BiP in PDAC cells. A Volcano diagram showing the differential genes by RUNX1 alteration based on TCGA dataset. B Spearman correlation analysis of RUNX1 and ER stress related markers: BiP, EIF2ΑK3(PERK), ERN1(IRE1α), and ATF6), based on the TCGA dataset. C Feature plot showing the co-expression relationship of RUNX1 and BiP expression in malignant ductal epithelial cells in the downloaded single-cell dataset (CRA001160), color ranges blue (low) to yellow (high)represents correlation score. D-E Representative images of BiP and RUNX1 staining (low and high) on successive slides of human PDAC tissues (D). The bubble diagram (E) showing expression of RUNX1 and BiP in each sample. The bubble size represents the number of cases. F Immunoblot analysis of RUNX1 and BiP expression in SW1990-RUNX1 and L3.7-shRUNX1 cells. G-H mRNA levels of RUNX1 and BiP in SW1990-RUNX1 and L3.7-shRUNX1cell lines by RT-qPCR. I ChIP analysis of SW1990-RUNX1 cells. Chromatin was immunoprecipitated using an anti-RUNX1 antibody and subjected to PCR. J SW1990-RUNX1 cells were transfected with a pGL3-BiP-widetype (wt), pGL3-BiP-mutation or pGL3-control vector. The results are presented as a fold-change Firefly activity relative to cells transfected with the control vector after normalization to Renilla activity. K The cell apoptosis of SW1990-RUNX1 cells transfected with siRNA targeting BiP(siBiP#1) by flowcytometry, under gemcitabine treatment (2 µM, 48 h). The average apoptosis rate in each group is shown in the column diagram. J Clonogenic assay of SW1990-RUNX1 cells and SW1990-vector cells transfected with siBiP#1, under gemcitabine treatment (200 nM). Colonies were stained with crystal violet (0.5%) after 14 d and counted using ImageJ software. Student’s t-test was used in the column diagram; scale bar, 200 µm; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig 3: Expression of cell cycle genes during BCR-mediated activation of Runx1 knockout B cells. (A) RNA-seq analysis of Runx1 c-k/o B cells showing upregulation and downregulation of genes in resting B cells (upper pie chart) and in 3-h anti-IgM activated B cells (lower pie chart) with respect to Cre-only control B cells. Three biological replicates were analyzed for each cell type. (B) GSEA of the RNA-seq data from 0- and 3-h anti-IgM + IL-4 activation of Runx1 c-k/o and Cre-only resting B cells. Values <0 in the graphs refer to genes whose expression is elevated in the Runx1 c-k/o B cells. (C) RT-qPCR analysis of expression of the Ccnd2 gene in resting B cells (top left panel) and after activation for the indicated times with anti-IgM + IL-4 (top right panel). Bottom panel, Western blot analysis of cyclin D2 protein levels after anti-IgM + IL-4 activation for the indicated times. Loading control: histone H3. (D) Single-cell analysis of the level of cyclin D2 protein in resting B cells and after 3 and 6 h of anti-IgM + IL-4 treatment using quantitative, automated single-cell imaging analysis (see Materials and Methods). Three independent experiments are shown. p values obtained using the Mann–Whitney U test are shown above each time point. (E and F) RT-qPCR analysis of cell cycle gene transcription (E) and immediate early gene transcription (F) in the Runx1 c-k/o B cells on activation with anti-IgM + IL-4 for 0–18 h. For (C), (E), and (F), values are mean ± SD. *p ≤ 0.5, **p ≤ 0.01, ***p ≤ 0.001, Student t test, n = 3. NES, normalized enrichment score; P, nominal p value.

Fig 4: Factor binding profiles of genes that are repressed by RUNX1. (A) Basemean values for expression of genes with functional roles in B cells that are upregulated in the Runx1 c-k/o B cells (see Table I). Values were obtained from the RNA-seq analysis at 0 and 3 h of anti-IgM + IL-4 treatment (see Supplemental Tables I and II). All values are the mean of three biological replicates. Adjusted p values for the differences in gene expression levels were <0.05 for at least one of the points. (B) ChIP-seq tracks showing the profiles of factor binding and histone modification in resting B cells of the genes shown in (A).

Fig 5: Status of Eμ enhancer in DP thymocytes. (A) Enhancer elements and transcription factors that bind to these sites are shown below the schematic of the DH-CH part of the IgH locus. Arrows originating from the enhancer represent bi-directionally transcribed eRNAs named as Iμ sense and Iμ antisense. (B) Enhancer-associated histone modifications were scored by ChIP using anti-H3K4me1 and anti-H3K27ac antibodies in CD19+ pro-B cells and DP thymocytes derived from TCRβ × Rag2−/− transgenic mice. Y-axis represents fold enrichment of the indicated amplicon in the immunoprecipitate compared to an equal amount of input DNA. For each independent experiment PCR was done in triplicate. The data shown is the mean of two independent ChIP experiments. Error bars represent standard error of the mean (n = 2). γ-actin was used as a positive control, TCF7 enhancer was used as a positive control for T-lineage-specific gene and Cγ3 was used as a negative control. (C) Levels of eRNAs that originate within the enhancer in DP thymocytes and pro-B cells. Reverse transcription was carried out with strand specific primers, or no primer (np), followed by amplification with primers that score for sense and antisense Iμ transcripts. RNA amounts were calculated based on a standard curve obtained from serial dilutions of 1 kb DNA spanning both sense and antisense transcribed region. (D) Transcription factor binding to Eμ was assayed by chromatin immunoprecipitation using antibodies directed against the indicated factors. Enrichment of specific amplicons in co-precipitated DNA was calculated relative to an equal amount of input DNA (Y-axis). Error bars represent standard error of the mean (n = 2). Positive controls for each transcription factor correspond to the first set of bars in each graph. IgG served as additional negative control. (E) YY1, E2A, Ets-1, and RUNX1 expression in DP thymocytes and Rag2−/− pro-B cells were assayed by immunoblotting with the respective antibodies.