Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder

Abstract

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.

Figure 1.

Nine (two published—D9, D10—and seven novel) ACD/MPV-causing microdeletions share a 75-kb putative regulatory region (SDR), mapping 257 kb upstream of FOXF1 (16q24.1). Array CGH plots of two deletions defining SDR are shown.

Figure 2.

Characterization of the SDR. (A) Segment 1 (∼10 kb; black rectangle) and Segment 2 (∼8 kb; red rectangle), harboring, among others, transcription factor binding sites and a part of the fetal lung–expressed lncRNA gene, are shown. The figure is drawn according to the 2009 Human Reference Sequence (GRCh37/hg19). (B) Insight into the ∼1.5-kb Fragment 1a showing (from top to bottom) the bisulfite sequenced region, methylation array oligo probes (red arrows) that indicate differential methylation, differentially methylated CpG island, ChIP-chip GLI2-binding region, 7× regulatory potential, mammalian evolutionary conservation, and CG content.

Figure 3.

Distant regulatory region, SDR, controls the activity of the FOXF1 promoter. (A,B) Results of the reporter assay experiments showing regulation of the FOXF1 promoter in HPMEC by fragments of Segments 1 and 2 of the SDR. (FOXF1p) FOXF1 promoter (0–5.5 kb upstream of ATG codon) cloned in pSEAP2Basic; (41.4delp) truncated FOXF1 promoter (0 to −3.0 kb, ACD case 41.4) cloned in pSEAP2Basic; (Seg1FOXF1p) distant upstream GLI-binding region (part of Fragment 1a) and FOXF1 promoter cloned in pSEAP2Basic; (Seg2>FOXF1p) Segment 2 putative enhancer and FOXF1 promoter cloned in pSEAP2Basic in the same orientation; Seg2<FOXF1p, the same enhancer in reverse orientation and FOXF1 promoter cloned in pSEAP2Basic; (vect) pGEM-T Easy vector; (Seg1) GLI-binding region of Fragment 1a cloned in pGEM-T Easy; (Seg2) putative enhancer of Segment 2 cloned in pGEM-T Easy. The presence of additional GLI2, constitutively expressed from the pCS2Gli2 vector, is denoted by +GLI2. The GLI-binding region of Fragment 1a stimulates activity of the FOXF1 promoter in cis (A) and in trans (B) settings. (C) Results of the 4C experiment revealing long-range physical interaction between the FOXF1 promoter and SDR. Coordinates of the SDR region interacting with the FOXF1 promoter (SDR-promoter interaction peaks) in HPMEC are (from top) 86,246,725–86,246,982 (0.05 < FDR ≤ 0.1) and 86,246,125–86,246,982 (0.1 < FDR ≤ 0.2). The two HPMEC panels represent results of independent experiments. No SDR-FOXF1 promoter interaction peak was detected in lymphoblasts, which do not express FOXF1.

Figure 4.

Methylation of the SDR CpG island decreases SDR potential to activate the FOXF1 promoter. (A) Methylation status of the FOXF1 promoter and SDR CpG island in normal fetal lung (a similar CpG methylation pattern was observed in DNA isolated from blood). Each lane represents a separate clone. (●) Methylated CpGs, (○) unmethylated CpGs. (Pink) Location of a cluster of GLI-binding site variants. (Red) Position of the consensus “core” GLI-binding site. Genomic coordinates of the bisulfite sequenced regions are: chr16:86,232,367–86,232,979 (SDR CpG island); chr16:86,542,223–86,542,807 (FOXF1 promoter 2); chr16:86,543,777–86,543,907 (FOXF1 promoter 1); chr16:6,544,458–86,545,037 (FOXF1 exon 1). (B) Dependence of the FOXF1 promoter activity on the methylation status of the SDR CpG island. The presence of additional GLI2 constitutively expressed from the pCS2Gli2 vector is denoted by +GLI2. (FOXF1p) FOXF1 promoter cloned in pSEAP2Basic; (vect) pGEM-T Easy; (Seg1) GLI-binding region (Fragment 1a) cloned in pGEM-T Easy; (mSeg1) GLI-binding region of Fragment 1a with in vitro–methylated CpG island cloned in pGEM-T Easy; (Seg1*) mock control for mSeg1.