The angiogenic function of endothelial cells is regulated by numerous mechanisms but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. Goethe-University researchers set out to identify novel and functionally important endothelial lncRNAs.
Epigenetically controlled lncRNAs in human umbilical vein endothelial cells (HUVEC) were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA Pulldown and Immunoprecipitation, Mass spectrometry, Micro-array, several knockdown approaches, CRIPSR-Cas9, Assay for Transposase-Accessible Chromatin-Sequencing and chromatin immunoprecipitation in HUVEC. Patient samples from lung and tumors were studied for MANTIS expression.
A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension (IPAH) and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet as well as in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with siRNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the SWI/SNF chromatin remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6 and COUP-TFII was regulated by ensuring efficient RNA Polymerase II machinery binding.
Model of mechanism of action for the lncRNA MANTIS
(Upper part) MANTIS lncRNA expression is controlled by the histone demethylase JARID1B. Due to the limited expression of MANTIS, BRG1 and BAF155 assembly is decreased, leading to more heterochromatin formation at the TSS of SOX18, SMAD6 and COUP-TFII, limiting RNA Pol II binding and transcription of those genes. (Lower part) In case of JARID1B knockdown, H3K4me3 levels arise at the TSS of MANTIS, allowing more MANTIS expression. MANTIS interacts nteracts with BRG1, allowing increased binding of BAF155, which leads to a higher ATPase activity of BRG1 and euchromatin formation at the TSS of SOX18, SMAD6 and COUP-TFII allowing RNA Pol II binding and thereby transcription of SOX18, SMAD6 and COUP-TFII, which leads to increased angiogenic function.