Transcriptional effects of INGAP-PP peptide in the control on the pancreatic beta-cell mass and function: A combined epigenetic and transcriptomic analysis

ROMERO, AGUSTÍN; HEIDENREICH, ANA C.; ROMAN, C.L.; ALGAÑARAS, M; GAGLIARDINO, JUAN J.; MAIZTEGUI, BÁRBARA; FLORES, LUIS E.; RODRÍGUEZ SEGUÍ, S.A.

Mar del Plata

Congreso; Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2022

Sociedad Argentina de Investigación Clínica (SAIC)

The Islet Neogenesis-Associated Protein pentadecapeptide (INGAP-PP) has been shown to improve β-cell mass and function1. In this talk I will present the bioinformatics approach that led us to elucidate some of the effect of this peptide on gene expression in an unbiased way. We performed RNA-seq analysis on rat pancreatic islets treated in vitro with INGAP-PP, and integrated these data with epigenetics profiles. First of all we validated the effect of the treatment by measuring glucose-stimulated insulin secretion. We identify 1669 differentially expressed genes by this treatment.(F1)We carried out functional annotation of the regulated genes. GO and GSEA reveals previously reported and novel signaling pathways potentially modulated by this peptide, such as Vegf, Pi3k, and Tgf-β, as well as biological processes including angiogenesis and ECM organization.(F2)In-depth analyses, integrating public pancreatic islet single-cell RNA-Seq2 data allowed us to find that some of the gene upregulated upon treatment are prominently expressed in minor cell type populations within pancreatic islets. This lead us to propose that INGAP-PP effects like angiogenesis and ECM organization may potentially be reached by interaction of non-endocrine cell, especially Stellate, Endothelial and Schwann cells.(F3)Then we integrated public ChIP-seq3 and ATAC-seq4 data profiled in the INS-1 cell line, (surrogate for rat β-cells). Interestingly, 1302 of these are already active in INS-1 (i.e. regions also enriched in H3K27ac). We also identified 25,803 enhancers with open chromatin (H3K4me1), of which 1362 were associated with the INGAP-PP regulated genes that had H3K4me3 signal in INS-1.(F4)An unsupervised k-means clustering of the INGAP-PP enhancers within a 6Kb window centered at the ATAC-seq summits revealed 3 distinct groups of enhancers differing in the H3K27ac signal (i.e. active regions): Active High, Active Low and Poised. This shows that a small subset of enhancer regions are already highly active in untreated INS-1 A differential de novo motif analysis revealed enrichments for transcription factor motifs matching STAT2/RUNX1, RARA/HNF4, HIF1 and MEF2 DNA binding sequences, among others.(F5)Finally we identified 75 previously unannotated rat transcripts which are upregulated by INGAP-PP. Integration of this data with the epigenomic analyses allowed us to discover 14 novel rat long-non coding RNAs which could have relevant functions in pancreatic islet cell biology (F6)