Long Non-coding RNA BGas Regulates the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

Cystic fibrosis (CF) is a life-shortening genetic disease. The root cause of CF is heritable recessive mutations that affect the cystic fibrosis transmembrance conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface. We show that CFTR is regulated transcriptionally by the actions of a novel long noncoding RNA (lncRNA), designated as BGas, that emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein coding sense strand. We find that BGas functions in concert with several proteins including HMGA1, HMGB1, and WIBG to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene and thereby affects transcription. Suppression of BGas or its associated proteins results in a gain of both CFTR expression and chloride ion function. The observations described here highlight a previously underappreciated mechanism of transcriptional control and suggest that BGas may serve as a therapeutic target for specifically activating expression of CFTR.

Figure 1

BGas and as4 mediated regulation of CFTR. (a) A schematic depicting the CFTR locus with transcriptional start sites (TSS) for CFTR, EST BG213071 (BGas), and those primers used to evaluate CFTR expression. The sasRNA target site (as4) in the BGas promoter is also shown along with the two exons making up BGas. (b) The effects of BGas over-expression using an exogenously expressed BGas (exBGas) on CFTR expression in 1HAEo- cells. The control (pcDNA3.1) and exBGas transfected cells are shown. (c,d) The effects of sasRNA as4 relative to control (pU6M2) on (c) BGas and (d) CFTR expression in 1HAEo- cells. (e,f) The effects of exBGas (e) and sasRNA as4 (f) on CFTR expression in CFPAC cells. (g) Dose-dependent effect of an siRNA targeting the as4 site (siRNA4) on ΔF508-CFTR chloride channel transport function in CF Human Bronchial Epithelial (CFhBE) primary cells (epithelial voltage clamp assay, Ussing chamber). (h) The localization of biotin containing BGas at the intergenic locus of CFTR in transfected CFPAC cells contrasted with the lambda biotin control. For b–f, the averages of triplicate treated cultures are shown with the standard error of the means and a P value from a paired T-test, *P < 0.05 and **P < 0.01.

Figure 2

The mechanism of BGas regulation of CFTR. BGas interacts specifically in the CFTR locus to modulate the binding of several proteins involved in DNA architecture and chromatin structure. (a) A close up snap-shot from UCSC genome browser of the as4 target site and those primers used to distinguish the epigenetic changes at the BGas targeted locus in intron 11 of CFTR. (b) The effects of over-expression of BGas using an exogenously expressed Bgas (exBGas) relative to control (pcDNA3.1) on H3K27me3 enrichment at BGas exon 1 (Set 8). (c,d) Relative enrichment of active RNAPII (serine 2, phospho 5) at the (c) BGas exon1/promoter (Set 7) or (d) BGas exon 1 (Set 8) in BGas transfected CFPAC cells. (e) Transcription is not required for BGas localization to CFTR. CFPAC cells were transfected with a biotin-labeled BGas transcript or Lambda transcript (control) (50 nmol/l) and then treated with alpha-amanitin. Biotin pulldowns were carried out 30 hours later to determine localization of the Biotin-BGas transcript. For b–e, the averages of triplicate treated cultures are shown with the standard error of the means and a P value from a paired T-test, *P < 0.05. (f) Determination of BGas associated proteins. Biotin-labeled oligonucleotides antisense to BGas (biotin-BG-1 and biotin-BG-2), were used to immunoprecipitate BGas in CFPAC cells. The eluates were subjected to LC/MS analysis and several candidates determined. Total spectrum counts for the top 10 candidates found in both biotin-BG-1 and biotin-BG-2 immunoprecipitations are shown. (g,h) Validation of LC/MS identified proteins by RNAi. (g) The expression of the mass spectrophotometry identified HMGA1, HMGB1, and WIBG was determined following suppression of each transcript with RNAi relative to a scrambled control siRNA (siCTRL) in non-CF 16HBE14o- cells that express high levels of endogenous CFTR. (h) The effects of knockdown of HMGB1, WIBG, and HMGA1 on CFTR expression. For (g–h), the averages of triplicate transfected 16HBE14o- cells are shown with the standard error of the means and p values from a paired two-sided T-test, *P < 0.05.

Figure 3

Model for BG213071 regulation of CFTR expression. (a) The CFTR locus is shown with the internal expressed BG213071 and truncated CFTR transcript NM_000492. (b) The BG213071 lncRNA (BGas) is expressed and localizes to the homology-containing locus in the CFTR gene body. The localization of BGas to it's target locus allows for chromatin structural and DNA binding proteins such as HMG-14, HMG-17, HMGB1, and WIBG to localize specifically to the CFTR gene body and affect the local structure of the gene ultimately diminishing RNAPII activity.