Splicing of a non-coding antisense transcript controls LEF1 gene expression.
Bottom Line: Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed.Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3.Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter.
Affiliation: Programa de Recerca en Càncer, Institut Hospital del Mar d'Investigacions Mèdiques, 08003 Barcelona, Spain UCL Cancer Institute, University College London, London, WC1E6BT, UK.Show MeSH
Related in: MedlinePlus
Mentions: The effect of the NAT on LEF1 mRNA expression was also determined by transfecting the spliced and unspliced forms of the transcript. We performed this analysis by two alternative experimental approaches. First, we transfected in vitro synthesized RNA corresponding to these NATs and checked LEF1 mRNA expression by strand-specific qRT-PCR. As shown in Figure 3A, the unspliced form down-regulated LEF1 mRNA whereas the spliced NAT did not have a significant effect. Co-transfection of the spliced NAT inhibited the action of the unspliced form, suggesting that the spliced NAT is acting as a dominant negative factor (Figure 3A). We also stably transfected a plasmid expressing the unspliced form of the transcript. As shown in Figure 3B (top), the unspliced NAT down-regulated LEF1 mRNA both in HT-29 M6 and RWP-1 Snail1 cells, without altering the stability of this RNA (see Supplementary Figure S3). A similar inhibition of LEF1 protein by unspliced NAT was also observed (Figure 3B bottom). Moreover, unspliced LEF1 NAT expression decreased the migration of both cell lines (Figure 3C) without affecting their growth rate (Supplementary Figure S4). Co-transfection of spliced NAT remarkably rescued the inhibition of migration caused by unpsliced NAT in RWP-1 Snail1 cells (Figure 3C). We did not detect a significant change in the phenotype of these cells. LEF1 NAT only caused a small increase in the mRNA levels of E-cadherin and other epithelial genes (Claudin4, Occludin) (Supplementary Figure S5A); E-cadherin up-regulation was also detected at protein level (Supplementary Figure S5B). No changes in the E-cadherin repressors Snail1 or Zeb1 (2,3) were observed, however ZEB2 RNA was significantly down-regulated (Supplementary Figure S5A), suggesting that LEF1 was contributing to the expression of this gene.
Affiliation: Programa de Recerca en Càncer, Institut Hospital del Mar d'Investigacions Mèdiques, 08003 Barcelona, Spain UCL Cancer Institute, University College London, London, WC1E6BT, UK.