Fig. 5: Identification of downstream targets of mH2A1.

A, B RNA sequencing (RNA‑seq) was performed on pancreatic cancer cells transfected with mH2A1 plasmids (Vector vs. mH2A1; four biological replicates, n = 4). Total RNA had an RIN value of ≥ 8.0 and concentrations sufficient for library preparation. Libraries were sequenced on an Illumina platform using paired‑end 150 bp reads, yielding approximately 4–6 × 10^7 reads per sample with a mean GC content of ~46%. Sequencing quality was high (> 98% of bases ≥ Q30), and the overall mapping rate to the GRCh38 reference genome exceeded 98%. Heatmap (A) shows the global expression patterns of differentially expressed genes upon mH2A1 overexpression; volcano plot (B) displays significance versus fold change (filter criteria: |Log2 FC | ≥ 1.5, P < 0.05). C, D ChIP‑seq was performed in pancreatic cancer cells to identify mH2A1 binding sites (one pair, n = 1). This study was conducted on the MGISEQ-T7 sequencing platform to perform ChIP-seq sequencing. A MGISEQ-T7 paired-end (PE) library with an insert size of approximately 300 bp was constructed and sequenced. Raw sequencing data were subjected to quality control and analyses of the ChIP-seq data. The total number of raw reads exceeded 80 million, with Q20 and Q30 values of > 99% and ≥ 97%, respectively, and an average GC content of ~45%. The effective rate was 99.9%. The heatmap depicts the distribution of mH2A1 peaks surrounding transcription start sites (TSS). E Distribution of mH2A1 binding sites across genomic functional regions. Identified binding sites were annotated to promoters, 5′ UTRs, exons, introns, 3′ UTRs, and intergenic regions; the chart displays the relative abundance of each category as a percentage. F The intersection of RNA‑seq and ChIP‑seq results revealed potential mH2A1 interaction targets. G IGV genome browser visualization of 17 putative mH2A1 binding sites at the TERT locus. The sites indicated by the blue boxes correspond to individual peaks and are significantly enriched relative to the input control (P < 0.05). H, I Multiplex immunofluorescence staining of UBE3A, mH2A1, and TERT in pancreatic tumor and adjacent normal tissues. Fluorescence intensities for the yellow (UBE3A), green (mH2A1), and red (TERT) channels were quantified using Image J. Each group included three independent biological replicates (n = 3); for each replicate, three random fields were acquired, and the mean of the three fields was used for analysis. Statistical comparisons were performed using a paired two‑tailed Student’s t‑test. J–M In two pancreatic cancer cell lines stably overexpressing or knocking down UBE3A, qRT‑PCR and Western blot were used to assess expression changes of UBE3A, mH2A1, and TERT. All experiments were independently repeated thrice (biological replicates, n = 3). Western blotting band intensities were quantified by densitometry and normalized to α‑Tubulin. Statistical comparisons were performed using a paired two‑tailed Student’s t‑test. All quantitative data are presented as mean ± SEM. Significance is indicated as **P < 0.01, ***P < 0.001, ****P < 0.0001.