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R5762 --> 484.10 --> Kras is one of the most frequently mutated genes in pancreatic cancer, with the G12D variant acting as a cancer driver mutation. Our previous work has integrated and defined gene networks induced by KrasG12D expression, particularly the expression of inflammatory gene clusters. Moreover, we observed that KrasG12D expression increases the overall protein levels of several histone marks, including H3K4me3, H3K27ac, H3K27me3, H3K9me3 and integrated data analysis correlated gene activation and silencing with the alteration of histone marks. Here we further consider KrasG12D‐driven changes in gene expression and hypothesize that G9a‐H3K9me2 may play an important role in the epigenomic regulation of this process. To investigate epigenomic mechanisms underlying KrasG12D‐induced gene expression networks, CUT&RUN technology was used to profile H3K4me3 (promoter), H3K9ac (promoter, enhancer), H3K27ac (enhancer, super‐enhancer), and H3K9me2 (silencer) histone marks at regulatory elements. We used a genetically engineered mouse model (GEMM)‐derived inducible KrasG12D cell line and compared the epigenetic changes after 24hr of oncogene induction. Data was analyzed by using Sparse Enrichment Analysis for CUT&RUN (SEACR). At the genomic level, we found most H3K4me3 peaks (~90%) were close to a transcription start site (TSS), and the number of H3K4me3 peaks were reduced ~9x after KrasG12D activation. However, for the gene silencing mark, H3K9me2, >80% peaks were located at intronic or inter‐genetic regions, and the number of peaks were increased >5x with KrasG12D. For H3K9ac and H3K27ac histone marks, we observed more peaks at intronic or inter‐genetic regions than at the TSS, indicating the exchange of histone marks at enhancer, super‐enhancer and silenced regions may play an important role in KrasG12D‐induced gene expression changes. We next focused on histone modifications at regulatory elements of inflammatory gene clusters, such as IL11, CXCL9‐CXCL10‐CXCL11, and IL4‐IL13 gene clusters. We further correlated epigenetic modifications with our gene expression data and found that the H3K4me3, H3K9ac, and H3K27ac, and H3K9me2 histone mark pathways are involved in the regulation of these inflammatory genes. Notably, these pathways are both, critical for pancreatic cancer development and potentially druggable, and therefore, these results bear both mechanistic and biomedical relevance to pancreatic carcinogenesis. [ABSTRACT FROM AUTHOR]

R5476 --> 484.8 --> Our laboratories study the role of the G9a‐H3K9me2 pathway in PDAC initiation and development in response to oncogenic KRASG12D mutation. Understanding how the epigenomic landscape responds to this mutation remains fundamental for the identification of potential mechanisms and targets for pancreatic oncogenesis. Unfortunately, because of its low levels in human cells and its discrete "block‐like" genome‐wide deposition, the H3K9me2 mark is more difficult to study than more abundant, sharp peak‐forming marks (e.g. H3K27ac). The aim of this study was to perform a comparison among multiple Next Generation Sequencing (NGS) technologies to identify the methodology with the best technical performance for detection of H3K9me2 at a genome‐wide scale. To do so, we performed Chromatin Immunoprecipitation (ChIP), CUT&RUN and CUT&TAG assays for the Histone H3 Lysine 9 di‐methyl histone mark (H3K9me2) followed by NGS on two pancreatic ductal epithelial‐like cells: hTERT‐HPNE E6/E7 and the KRASG12D mutant‐expressing hTERT‐HPNE E6/E7 KRASG12D cells. As expected, ChIP‐Seq required the largest number of total reads for peak detection. Both ChIP‐Seq and CUT&TAG had the highest percentage of high‐quality reads (>75%), while CUT&RUN performed below the 50% mark. No significant differences in library size were observed among all three techniques. Overall, CUT&TAG allowed identification of the largest number of H3K9me2‐marked genes in parental HPNE and KRASG12D‐expressing HPNE cells. Spearman correlation values above 0.8 for inter‐sample comparison indicated a high degree of repeatability for CUT&TAG replicates. In addition, CUT&TAG showed the largest occupation of the H3K9me2 mark near the transcription start site (TSS) of identified genes. Therefore, we have compared three methodologies for the evaluation of H3K9me2 occupation at the genome‐wide scale and identified CUT&TAG as the method with the highest quality, lowest noise‐to‐signal ratio, and highest repeatability. In conclusion, CUT&TAG represents the best option for NGS of the H3K9me2 histone mark in pancreatic cells. Notably, because this methodology is the best among the three tested for miniaturization of epigenomic studies, this information bears relevance to translational research in human, where sample availability is limited. [ABSTRACT FROM AUTHOR]