LETR1 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C

Abstract

Recent studies have revealed the importance of long noncoding RNAs (lncRNAs) as tissue-specific regulators of gene expression. There is ample evidence that distinct types of vasculature undergo tight transcriptional control to preserve their structure, identity, and functions. We determined, for the first time, the global lineage-specific lncRNAome of human dermal blood and lymphatic endothelial cells (BECs and LECs), combining RNA-Seq and CAGE-Seq. A subsequent genome-wide antisense oligonucleotide-knockdown screen of a robust set of BEC-and LEC-specific lncRNAs identified LESR2 as a critical gatekeeper of the global LEC transcriptome. Deep RNA-DNA, RNA-protein, and phenotype rescue analyses revealed that LESR2 acts as a nuclear trans-acting lncRNA modulating, via key epigenetic factors, the expression of essential target genes, including KLF4 and SEMA3C, governing the growth and migratory ability of LECs. Together, our study provides new evidence supporting the intriguing concept that every cell type expresses precise lncRNA signatures to control lineage-specific regulatory programs.The blood and lymphatic vascular systems are essential for the efficient transport of oxygen, nutrients, signaling molecules, and leukocytes to and from peripheral tissues, the removal of waste products, and the preservation of fluid homeostasis. Increased activation or impaired function of these vascular networks represent a hallmark of many pathological conditions, including cancer progression, chronic inflammatory diseases, and diseases leading to blindness 1-3 .During development, the blood vascular system arises from endothelial cell progenitors that differentiate from mesodermal cells, mostly through the expression of the transcription factor (TF) ETV2. Activation of the VEGFA/VEGFR2 signaling and expression of blood vascular endothelial cell (BEC) markers, such as NRP1 and EphrinB2, further differentiate these precursor cells into BECs, which then form the hierarchical network of blood vessels 4 . In contrast, lymphatic vasculogenesis starts after the establishment of the blood circulatory system. Thereafter, a distinct subpopulation of endothelial cells lining the cardinal vein starts differentiating by expressing the TF PROX1, the master regulator of lymphatic endothelial cell (LEC) identity, via the TFs SOX18 and COUPTFII. Once exiting the veins, LECs starts expressing other lymphatic-specific markers, such as podoplanin, VEGFR3, and NRP2, and they migrate, in a VEGFC-dependent manner, to form the primary lymph sacs from which the lymphatic vascular system further develops following sprouting, branching, proliferation, and remodeling processes 5 . However, a nonvenous origin of LECs has also been described in the skin, mesenteries, and heart 6-8 . In adulthood, while the blood and lymphatic vasculature are generally quiescent, they can be readily activated in pathological conditions such as wound healing, inflammation, and cancer by disturbance of the natural balance of pro-and anti-(lymph)angiogenic factors 1,9 . Therefor...

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function

“…This genome-wide function of LETR1 results in the control of the growth and migration of LECs. This study highlights the high potential of lncRNAs to regulate lymphatic-specific cellular mechanisms (Ducoli et al, 2020) (Figure 3C). Overall, a large number of studies have greatly deepened our understanding of how LEC gene expression is regulated to establish and maintain a mature functional state.…”

“…The implementation of model organisms (e.g., mice, chicken, and zebrafish) suitable to study lymphatic biology represents a great opportunity to quickly increase the Review knowledge about the function of these newly discovered lymphangiogenic regulatory mechanisms. Along these lines, recent technological advances in sequencing and genome-wide precipitation methods have provided additional pieces of evidence for the role of genomewide epigenetic modifications and lncRNAs in the establishment and maintenance of LEC identity and function (Ducoli et al, 2020;Tacconi et al, 2020). During the course of our studies of the LEC-specific lncRNA LETR1, we found that the usage of antisense oligonucleotides (ASOs) to target a gene of interest specifically expressed in LECs represents a promising and efficient approach.…”

Common and distinct functions of mouse Dot1l in the regulation of endothelial transcriptome

“…C-X-C motif chemokine ligand 10, one of the upregulated genes in OE-Dot1l BECs and LECs, promotes the migration of cardiac microvascular ECs (CMEC), likely by regulating the p38/focal adhesion kinase pathways without changing CMEC proliferation and viability (Xia et al, 2016). A recent study showed that LETR1, an LEC-dominant long noncoding RNA, modulates the expression of semaphorin 3C, one of the upregulated genes in OE-Dot1l BECs and LECs, and promotes the growth and migration of LECs by changing chromatin structure (Ducoli et al, 2021). Williams et al have performed a series of siRNA screening assays by targeting individual proteincoding genes and identified 111 genes critical for BEC and/or LEC migration (Williams et al, 2017).…”