Abstract 14714: The Antisense Transcript of GATA6 Interacts With the H3K4me3 Deaminase LOXL2 and Epigenetically Regulates Endothelial Gene Expression
Recent studies suggest that more than 80% of the human genome is transcribed, however, the majority of all transcripts is non-coding. Although the role of distinct non-coding RNAs is well-defined, little is known about the function of long non-coding RNAs (lncRNAs). Current research suggests a broad functional spectrum, e.g. miRNA-sponging, modulation of mRNA splicing and stability, or recruitment of chromatin modifying enzymes. Regarding endothelial cells, the response to hypoxia and the regulation of angiogenic activity are key events in the context of several diseases, however, the involvement of regulatory lncRNAs and their molecular function is not conclusively defined. By using RNA-seq and qRT-PCR, we identified and validated the lncRNA GATA6-AS to be significantly up-regulated by hypoxia in HUVECs (24h; 0.2% O2). Functional studies revealed that GapmeR-mediated silencing of GATA6-AS strongly impairs sprouting angiogenesis in vitro (to 50% ± 8%; p≤0.05) and ex vivo (to 58% ± 3%; p≤0.05) in mouse aortic ring assays. Subcellular fractionation showed a predominant nuclear localization of GATA6-AS. Therefore, we characterized GATA6-AS in more detail: First, by using antisense affinity purification and mass spectrometry, we identified the chromatin modifier LOXL2 (H3K4me3 deaminase) as GATA6-AS-associated protein and confirmed this interaction by RNA immunoprecipitation and in vitro pulldown assays. Second, gene expression profiling identified ~71% of GATA6-AS-regulated genes to be inversely regulated by LOXL2 silencing, including numerous angiogenesis-related genes, e.g. ICAM1. Third, GATA6-AS silencing lead to significantly increased co-IP efficiencies of the ICAM1 promoter in LOXL2 chromatin immunoprecipitations (313% ± 81%; p≤0.05) and in parallel reduced global H3K4me3 levels, compared to controls. Taken together, these results strongly argue for GATA6-AS acting as negative regulator of nuclear LOXL2. In summary, we characterized GATA6-AS as novel hypoxia-induced lncRNA and provide mechanistic insights into the epigenetic regulation of endothelial gene expression and sprouting angiogenesis by the interplay between the chromatin modifying enzyme LOXL2 and its negative regulator GATA6-AS.
Author Disclosures: N. Jaé: None. P. Neumann: None. D. John: None. S. Uchida: None. M. Krüger: None. S. Dimmeler: None.
- © 2016 by American Heart Association, Inc.