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      Ultra-conserved non-coding sequences within the FOXF1 enhancer are critical for human lung development

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          Abstract

          Heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions, involving the mesenchymal forkhead box family transcription factor gene, FOXF1, or its distant lung-specific enhancer, are responsible for 80%–90% of cases of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). 1 ACDMPV is a lethal lung developmental disorder with severe progressive respiratory failure and persistent pulmonary arterial hypertension (Supplemental Material). Intriguingly, in contrast to point mutations in FOXF1, the ACDMPV-causative CNV deletions arise de novo almost exclusively on the maternal chromosome 16q24.1. Thus far, we and others have described 50 de novo CNV deletions that arose on maternal chromosome 16 and only three de novo CNV deletions that arose on paternal chromosome 16q24.1 (Fig. S1). Here, we define an ∼660 bp ultra-conserved non-coding interval (660UCR) within the FOXF1 enhancer as critical for FOXF1 cis-regulation and normal human lung development. Heterozygous loss of this region on paternal chromosome 16 was found causative for ACDMPV. We also describe a novel enhancer lncRNA gene, RP11-805I24.3, located in the proximity to 660UCR and overlapping another ∼1 kb ultra-conserved interval (1000UCR), as essential for the FOXF1 expression. Based on the obtained data, we propose a bimodal structure and parental functional dimorphism of the FOXF1 enhancer. The lung-specific FOXF1 enhancer in humans has a complex structure, consisting of the proximal Unit 1 and distal Unit 2 (Fig. 1A). 1 , 2 Both units encode regulatory lung-expressed lncRNAs and feature parent-of-origin-specific epigenetic modifications. Using genome sequencing in ACDMPV family trio (204.1–3), we have identified a pathogenic heterozygous, 8.8 kb CNV deletion within Unit 1 that arose de novo on the paternal chr16q24.1 (Fig. 1A, S2–S6). Together with 33 other CNV deletions of the FOXF1 enhancer, this second smallest deletion enabled identification of the ultra-conserved 660UCR interval. Longer stretches of homology with 660UCR can be found in the genomes of lobe-finned fishes, including coelacanth and lungfish, whereas short sequence stretches are visible in some actinopterygian fishes with the vascularized swim bladder (Fig. 1A). For comparison, Unit 2 of the enhancer contains two non-coding intervals which are conserved down to turtles only. Figure 1 Bimodal structure and parental dimorphism of the ∼60 kb FOXF1/FENDRR lung-specific enhancer. (A) UCSC genome browser representation of the enhancer, defined by the smallest deletion overlap of 29 pathogenic CNV deletions at chromosome 16q24.1 leaving FOXF1 intact (Fig. S1). On the top are four additional different-sized CNV deletions identified in ACDMPV patients on maternal (red) or paternal (blue) chromosome 16q24.1 that provided further insight into bimodal structure and function of the enhancer. Blue arrows in Unit 1 indicate an ∼660 bp ultra-conserved interval, 660UCR, mapping within the ∼4 kb smallest deletion overlap of 33 CNV deletions, and antiparallel pair of the lung-expressed lncRNA genes LINC01082 and RP11-805I24.3 overlapping another ultra-conserved interval, 1000UCR. The red arrows in Unit 2 indicate (i) the lung-specific lncRNA LINC01081 gene (ii) the interval with the typical epigenetic signatures of an enhancer, and (iii) two differential methylation regions described recently by Slot and coworkers. 3 In addition, our ChIP-seq studies showed TBX4 binding to the Unit 2 (yellow rectangle). Black arrows indicate coelacanth and spotted gar fish. At the bottom, there are five benign CNV deletions assessed from DGV, involving Unit 2 of the enhancer, identified in apparently healthy individuals. We suggest these polymorphic deletions may be located on paternal chromosome 16q24.1. (B) Luciferase reporter assay showing the significant increase of FOXF1 promoter activity by juxtaposed fragment of the enhancer Unit 1 containing the ultra-conserved 660UCR interval. (C) Knock-down experiments in IMR-90 cells showing regulatory relationships between enhancer lncRNAs, FENDRR, and FOXF1 (P ≤ 10−n (n = 2∗–4∗) and <0.05 (∗)). (D) Knock-in of LINC01082 showing suppression of FOXF1 by overexpression of this lncRNA. (E) The analysis of the methylation status of the ultra-conserved 1000UCR interval of the FOXF1 enhancer mapping within the overlapping pair of mutually antisense lncRNA genes LINC01082 and RP11-805I24.3. ACDMPV lung (179.3, 28.3, 60.4, 125.3, 155.3, 170.3), blood (204.3, 205.3) and umbilical cord (180.3) tissues were analyzed. PCR was done using undigested (u) and digested (c) DNA. Figure 1 Applying luc2 reporter assay in fetal lung fibroblasts IMR-90, we have shown that the 660UCR interval increases the activity of the FOXF1 promoter two-fold (Fig. 1B), consistent with the fact that the CNV deletions of the paternal 660UCR allele in pts 204.3 and previously reported 122.3 (Fig. S1) led to full ACDMPV phenotype. A portion (EH38E1835120) of this interval is annotated in ENCODE's Candidate Cis-Regulatory Elements (cCREs) database as having distal enhancer-like signature. In addition, mouse region syntenic to human 660UCR was shown to function as the Foxf1 enhancer. 3 The second ultra-conserved region of Unit 1, 1000UCR, is included in GeneHancer regulatory element GH16J086193 and overlaps the lncRNA genes LINC01082 and RP11-805I24.3. Knock-down of these lncRNAs using siRNA in IMR-90 cells, showed that RP11-805I24.3 positively regulates expression of FOXF1, FENDRR, and other analyzed here lncRNAs (Fig. 1C), suggesting that it may function as a general transcriptional activator. On the other hand, LINC01082 (Fig. S7), which has expression of an order of magnitude lower than that of RP11-805I24.3, decreases expression of FOXF1 when up-regulated in IMR-90 cells from the pcDNA-based construct to the level of RP11-805I24.3 (Fig. 1D). Importantly, both 660UCR and 1000UCR bind EP300, a histone acetyltransferase that catalyzes H3K27ac deposition typical for active enhancers and partially overlap the RNA PolII binding site (ENCODE's ChIP-seq database). Moreover, EP300 is known to directly interact with HIF1A in regulation of hypoxia-induced VEGF and with RNA PolII (GeneCards). Using luc2 reporter assay, we have also found that the 660UCR interval does not regulate RP11-805I24.3 promoter (Fig. S8). Thus, 660UCR and RP11-805I24.3 seem to positively regulate FOXF1 expression independently of each other. Applying siRNA knock-down to FOXF1, we sought whether FOXF1 controls expression of its enhancer lncRNAs in a manner similar to how it regulates FENDRR. We have found that FOXF1 positively regulated LINC01082 but not RP11-805I24.3 (Fig. 1C). Thus, there is no evidence for a regulatory feedback loop interaction between FOXF1 and lncRNAs encoded in its enhancer. Interestingly, Unit 1 of the FOXF1 enhancer was previously shown to be differentially methylated at CpG dinucleotides. 2 , 4 Here, using methylation sensitive digestion assay, we have found methylation of paternal but not maternal allele of 1000UCR (Fig. 1E), whereas both parental alleles of the 660UCR were methylated, although showing a trend towards stronger methylation of the paternal allele (Fig. S9). Unit 2 features lung-specific epigenetic histone 3 modifications typical of an active enhancer, and includes the 3′ portion of the lung-expressed lncRNA gene LINC01081 (Fig. 1A). In this ∼5 kb-large interval, we have described previously four rare non-coding SNVs mapping in trans to heterozygous pathogenic SNV and CNV deletions involving FOXF1 and/or its enhancer, that, likely acting as hypermorphs, might have ameliorated the lethal ACDMPV phenotype. Interestingly, Unit 2 was also reported to exhibit features of differential CpG methylation (SNP rs1621902) 2 and partially overlaps ChIP-seq-determined TBX4 binding region (Fig. 1A). Moreover, it features putative allelic differences of the H3K27Ac profile and the presence of two differentially methylated regions (Fig. 1A). Using methylation-sensitive restriction digestion, we have determined that the region neighboring rs1621902 is methylated stronger on the paternal chr16q24.1 (Fig. S9). Previously, we have proposed that FOXF1 locus may be responsible for key features of the maternal uniparental disomy 16, UPD(16), phenotype. 5 In contrast to phenotypically benign paternal UPD(16), patients with maternal UPD(16) sometimes manifest features observed in ACDMPV, including heart defects, pulmonary arterial hypertension, tracheoesophageal fistula, gut malrotation, absent gallbladder, renal agenesis, hydronephrosis, imperforate anus, and single umbilical artery. 1 This suggests that the stronger paternal enhancer may also act more ubiquitously than its maternal allele which may be more lung-specific. Corroboratively, the full lethal ACDMPV phenotype associated with CNV deletions on paternal chr16q24.1 in patients 122.3 and 204.3 and milder ACDMPV phenotype in the longer surviving patient 99.3 (who had lung transplantation) with a larger-sized CNV deletion on maternal chr16q24.1 (Fig. 1, S1) suggest that Unit 1 may act as a stronger lung enhancer on paternal chr16q24.1. According to the “seesaw” mechanism, higher methylation level of the 660UCR on paternal chr16q24.1 would favor binding of enhancerous EP300 through the exclusion of H3K4me3 methylases, whereas lower methylation of maternal allele of the LINC01082 regulatory interval in Unit 1 might increase expression of LINC01082 and thus suppress FOXF1. In contrast, the association of CNV deletion on maternal chromosome 16q24.1 in patient 144.3 (Fig. 1, S1) with full ACDMPV implies Unit 2 acting as a stronger lung enhancer on maternal chromosome 16q24.1. In conclusion, we propose that two ultra-conserved intervals of the FOXF1 enhancer, 660UCR and 1000UCR, the latter overlapping the lncRNA genes LINC01082 and RP11-805I24.3, form Unit 1 that plays an essential role in human lung development. We propose that this unit acts as a stronger FOXF1/FENDRR enhancer on the paternal copy of chromosome 16q24.1 whereas Unit 2, with the typical chromatin epigenetic features of an active enhancer, may play a modifier role and act as a stronger lung enhancer on the maternal copy of chromosome 16q24.1. Moreover, the ultra-conserved non-coding enhancer intervals, especially those within Unit 1, can be traced down to lungfish, coelacanth (sarcopterygians) and even spotted gar (actinopterygians), suggesting that their appearance in fishes might have been one of important steps in lung evolution. Author contributions PSz and EYB, performed the molecular experiments, TM, TG, and JAK performed the computational analyzes, MB collected the clinical data, NC-S and GA evaluated the histopathological specimen, PSz, DW, and PSt analyzed and interpreted the molecular data, PSz and PSt designed the study concept, wrote, and critically revised the final version of the article. Conflict of interests The authors declare no conflicts of interest. Funding This work was supported by the grants awarded by the US 10.13039/100000002 National Institutes of Health (NIH), 10.13039/100000050 National Heart Lung and Blood Institute (NHLBI) R01HL137203 and 10.13039/100009633 Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) grant R01HD087292 to P.St.

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          Most cited references5

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

          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.
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            Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations

            Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.
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              Long-range enhancers modulate Foxf1 transcription in blood vessels of pulmonary vascular network.

              Intimate crosstalk occurs between the pulmonary epithelium and the vascular network during lung development. The transcription factor forkhead box f1 (Foxf1) is expressed in the lung mesenchyme and plays an indispensable role in pulmonary angiogenesis. Sonic hedgehog (Shh), a signalling molecule, is expressed in lung epithelium and is required to establish proper angiogenesis. It has been suggested that Foxf1, a downstream target of the Shh signalling pathway, mediates interaction between angiogenesis and the epithelium in lung. However, there has been no clear evidence showing the mechanism how Foxf1 is regulated by Shh signalling pathway during lung development. In this study, we investigated the lung-specific enhancers of Foxf1 and the Gli binding on the enhancers. At first, we found three evolutionarily conserved Foxf1 enhancers, two of which were long-range enhancers. Of the long-range enhancers, one demonstrated tissue-specific activity in the proximal and distal pulmonary blood vessels, while the other one demonstrated activity only in distal blood vessels. At analogous positions in human, these long-range enhancers were included in a regulatory region that was reportedly repeatedly deleted in alveolar capillary dysplasia with misalignment of pulmonary vein patients, which indicates the importance of these enhancers in pulmonary blood vessel formation. We also determined that Gli increased the activity of one of these long-range enhancers, which was specific to distal blood vessel, suggesting that Shh regulates Foxf1 transcription in pulmonary distal blood vessel formation.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                18 May 2022
                November 2022
                18 May 2022
                : 9
                : 6
                : 1423-1426
                Affiliations
                [a ]Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
                [b ]Institute of Computer Science, Warsaw University of Technology, Warsaw 00-665, Poland
                [c ]Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, Poznan 60-806, Poland
                [d ]Department of Pathology, Texas Children's Hospital, Houston, TX 77030, USA
                [e ]Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 77030, USA
                [f ]Department of Anaesthesia, Kepler University Medical Campus III, Linz 4020, Austria
                [g ]Department of Pathology, Medical University of Vienna, Vienna 1090, Austria
                [h ]Department of Medical Genetics, Kepler University Hospital Med Campus IV, Johannes Kepler University Linz, Linz 4020, Austria
                Author notes
                []Corresponding author. Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, Rm ABBR-R809, Houston, TX 77030, USA. pawels@ 123456bcm.edu
                Article
                S2352-3042(22)00136-2
                10.1016/j.gendis.2022.05.002
                9485266
                36157490
                d420af57-761f-4b40-afd4-2dfa977ff376
                © 2022 Chongqing Medical University. Production and hosting by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 16 February 2022
                : 26 April 2022
                : 1 May 2022
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