PMCID string | Title string | Sentences string |
|---|---|---|
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Source data are provided as a Source Data file. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We found that the length of the element tested, and whether it matched the extent of open chromatin of an endogenous enhancer, affected a SNP’s direction of effect in reporter assays. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | First, for the two SNPs with opposite directions of effect in MPRA and luciferase reporter assays (FOXE1 24 kb and rs201265), we repeated the latter using shorter elements that matched the lengths used in the MPRA (i.e., 161 bp). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In these luciferase reporter assays, the directions of effect concorded with those from the MPRA (Supplementary Fig. 10a and Supplementary Data 12). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Second, for three promising SNPs (IRF6 −10 kb, IRF6 −22 kb and FOXE1 24 kb) with allele-specific effects in the MPRA and in the luciferase reporter assays, we repeated the latter using slightly longer elements (936 bp to 1.1 kb) that aligned with open chromatin (i.e., ATAC-seq peaks) in HIOEC and in NHEK (Supplementary Fig. 10b–d and Supplementary Data 10). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Remarkably, for all three SNPs the directions of effect were reversed relative to those using 701 bp elements: in the assays with the longer elements, the risk alleles of IRF6 −10 kb and IRF6 −22 kb SNPs reduced the activity, and the risk allele of FOXE1 24 kb elevated the activity, of the enhancers in which they reside (Supplementary Fig. 10b–d). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To summarize, in reporter assays, the length of the element tested affected a SNP’s direction of effect but not whether there was an effect. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | At this point in the study, we conducted RNA-seq on our stock of GMSM-K cells and compared expression levels of epithelial and mesenchymal markers to those in primary adult keratinocytes (NHEK) and in human embryonic palatal mesenchymal (HEPM) cells (Supplementary Data 13 and Supplementary Fig. 11a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | As expected, GMSM-K expressed higher levels of oral epithelium markers PITX1 and FOXE1 transcripts than either NHEK or HEPM, consistent with their origin from fetal oral keratinocytes. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In addition, they expressed high levels of epithelial markers KRT8 and KRT18 and low levels of mesenchymal markers VIM and CDH2, similar to NHEK and unlike HEPM. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | However, unexpectedly, they expressed much lower levels of the epithelial marker CDH1 and keratinocyte markers KRT14, TP63, and IRF6 than NHEK did; we confirmed three of these markers with immunostaining (Supplementary Fig. 11a, b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We concluded that our stock of GMSM-K had lost robust epithelial features without fully converting to a mesenchymal phenotype. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Therefore, we repeated the luciferase reporter assays in primary neonatal keratinocytes (HEKn). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We found that, again, the results concorded with those from the MPRA at 11 of 13 SNPs regarding whether the SNP had allele-specific effects on activity (Fig. 3b–d and Supplementary Fig. 12). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | In summary, although our stock of GMSM-K lacks robust epithelial features, the results from an MPRA conducted in GMSM-K and those from luciferase reporter studies in HEKn largely concorded. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To test whether the IRF6 −10 kb and IRF6 −22 kb SNPs affect enhancer activity in their native genomic contexts we engineered the genotype of WTC-11 induced pluripotent stem cells (iPSCs), which are heterozygous for both SNPs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | For each SNP, we generated two clones each that were homozygous for the risk allele or for the non-risk allele (Supplementary Figs. 13a, b, 14a, b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Next, we subjected three replicates of each clone to a 10-day protocol that induces iPSCs to differentiate into embryonic oral epithelial cells (induced oral epithelial cells, iOE) (Supplementary Fig. 13a, b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Quantitative RT-PCR (qRT-PCR) revealed that the average level of IRF6 expression in cells homozygous for the risk allele of either SNP (and heterozygous at the other) was lower than that in clones homozygous for the non-risk allele of that SNP (Fig. 3e, f), consistent with the luciferase reporter assays using the longest elements. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We also engineered the genotype of these SNPs in GMSM-K cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Despite the much lower expression of IRF6 in GMSM-K compared to in iOE cells, again, cells homozygous for risk alleles had lower IRF6 expression than cells homozygous for non-risk alleles (Supplementary Fig. 15a–f). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These results indicate that the IRF6 −10 kb SNP and the IRF6 −22 kb SNP both directly affect IRF6 expression. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Similarly, we engineered the genotype of the FOXE1 24 kb SNP. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The parental iPSCs were heterozygous, and we engineered three clones each to be homozygous for the risk allele or the non-risk allele and differentiated them in triplicate into iOE cells (Supplementary Fig. 16a, b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | qRT-PCR revealed that the average level of FOXE1 in cells homozygous for the risk allele was higher than that of the homozygous for the non-risk allele (Fig. 3g), consistent with the luciferase reporter assays using the longest elements, indicating that the FOXE1 24 kb SNP directly affects FOXE1 expression. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Using an online tool we predicted that the risk allele of the IRF6 −10 kb SNP diminishes the affinity for two transcription factors (i.e., AR and SOX10) and elevated it for POU5F1B plus sixteen members of the FOX family, including FOXE1 (JASPAR score for FOXE1 site MA1487.1: risk, 14.1; non-risk, 8.6) (Fig. 4a and Supplementary Data 14, 15). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Members of the FOX family can function as transcriptional activators or repressors. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Mutations in FOXE1 cause Bamforth–Lazarus syndrome and SNPs near it are associated with risk for non-syndromic OFC. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Using an antibody against FOXE1, we conducted ChIP in iOE cells generated from the parental iPSCs. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | qPCR revealed more chromatin precipitated at the IRF6 −10 kb SNP locus by anti-FOXE1, and by anti-H3K27Ac, than at an intergenic region, consistent with the SNP being in an enhancer bound by FOXE1 (Fig. 4b, c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | While the iOE cells were heterozygous at this SNP, like the parental iPSCs from which they were derived (Fig. 4d), PCR and sequencing revealed that chromatin precipitated by the anti-FOXE1 antibody was enriched for the risk allele (Fig. 4d, two additional replicates in Supplementary Fig. 17a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | By contrast, the anti-H3K27Ac antibody pulled down more of the non-risk allele (Fig. 4d and Supplementary Fig. 17a), consistent with higher activity of the enhancer and higher expression of IRF6 in cells with the non-risk genotype. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Similarly, anti-FOXE1 ChIP in GMSM-K homozygous for the risk or non-risk allele of IRF6 −10 kb SNP revealed stronger binding of FOXE1 to the risk allele (Supplementary Fig. 18a). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These results indicate that the IRF6 −10 kb SNP risk allele favors the binding of FOXE1.Fig. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 4Risk allele of IRF6 -10 kb SNP (rs11119348) promotes binding of transcriptional repressor FOXE1.a Consensus FOXE1 binding motif from the JASPAR database of transcription factor DNA-binding preferences (Matrix ID: MA1847.1) and alignment of the variant site in several mammals. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele (A) is the reference allele and has a higher frequency than the non-risk allele (C) in most populations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | b, c Percent input identified by ChIP-qPCR for anti-FOXE1 and anti-H3K27Ac, respectively, in iOE cells heterozygous for rs11119348 using primers specific to the IRF6 -10 kb enhancer site or, as a negative control, to a region 103.7 kb upstream of the IRF6 transcription start site that lacked ATAC-Seq and H3K27Ac ChIP-Seq signals in HIOEC or NHEK. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Error bars refer to three ChIP replicates and expressed as mean ± SD. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | d Sequencing of anti-FOXE1 and anti-H3K27Ac ChIP-PCR products from cells heterozygous for rs11119348 using the indicated antibody. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | e Scattered dot plot of relative luciferase activity of the IRF6 -10 kb reporter construct (longest version) harboring the risk allele of rs11119348, in wildtype (WT) or a clone of FOXE1 homozygous knockout (KO) GMSM-K cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | f Scattered dot plot of relative levels of IRF6 mRNA in WT and FOXE1-KO GMSM-K cells assessed by qRT-PCR. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels of IRF6 are normalized against ACTB. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | g Model showing binding of FOXE1 to the IRF6 -10 kb enhancer, which is favored by the risk allele and which reduces IRF6 expression. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Source data are provided as a Source Data file. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | a Consensus FOXE1 binding motif from the JASPAR database of transcription factor DNA-binding preferences (Matrix ID: MA1847.1) and alignment of the variant site in several mammals. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele (A) is the reference allele and has a higher frequency than the non-risk allele (C) in most populations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | b, c Percent input identified by ChIP-qPCR for anti-FOXE1 and anti-H3K27Ac, respectively, in iOE cells heterozygous for rs11119348 using primers specific to the IRF6 -10 kb enhancer site or, as a negative control, to a region 103.7 kb upstream of the IRF6 transcription start site that lacked ATAC-Seq and H3K27Ac ChIP-Seq signals in HIOEC or NHEK. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Error bars refer to three ChIP replicates and expressed as mean ± SD. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | d Sequencing of anti-FOXE1 and anti-H3K27Ac ChIP-PCR products from cells heterozygous for rs11119348 using the indicated antibody. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | e Scattered dot plot of relative luciferase activity of the IRF6 -10 kb reporter construct (longest version) harboring the risk allele of rs11119348, in wildtype (WT) or a clone of FOXE1 homozygous knockout (KO) GMSM-K cells. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | f Scattered dot plot of relative levels of IRF6 mRNA in WT and FOXE1-KO GMSM-K cells assessed by qRT-PCR. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Expression levels of IRF6 are normalized against ACTB. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | g Model showing binding of FOXE1 to the IRF6 -10 kb enhancer, which is favored by the risk allele and which reduces IRF6 expression. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Source data are provided as a Source Data file. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | To test the importance of FOXE1 in regulating the IRF6 −10 kb enhancer we repeated the luciferase reporter assays using the IRF6 −10 kb enhancer (longest version) harboring the risk allele of the IRF6 −10 kb SNP, and compared the results between parental GMSM-K and those in which we had deleted the FOXE1 using CRISPR/Cas9 reagents (Supplementary Figs. 18b, 19). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The FOXE1 knockout cells were clonal isolates and therefore entirely or mostly pure for the loss of FOXE1. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We chose GMSM-K over HEKn because the expression of FOXE1 is higher in GMSM-K than in HEKn (Supplementary Fig. 11c). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | GMSM-K are homozygous risk for the IRF6 −10 kb SNP, where FOXE1 binds more avidly. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Interestingly, in cells lacking FOXE1 the activity of the IRF6 −10 kb reporter was higher than in the control cells (Fig. 4e). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We saw analogous results in cells transfected with siRNA targeting FOXE1 or a non-targeting siRNA (Supplementary Fig. 18c, d). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Moreover, levels of IRF6 expression were higher in FOXE1-knockout or FOXE1 knockdown GMSM-K than in the respective controls (Fig. 4f and Supplementary Fig. 18e). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | While the binding of other transcription factors may be affected by the allele of the IRF6 −10 kb SNP, these results suggest its effect on binding of FOXE1 is sufficient to alter the activity of this enhancer (Fig. 4g). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele of the IRF6 -22 kb SNP diminished the predicted affinity for two transcription factors (GATA2 and HSF4) and elevated it for NFKB2, ZNF454, and ten members of the ETS family, including ETS2 (JASPAR score for ETS2 site MA1484.1, risk: 4.9; non-risk, -4.1) (Fig. 5a and Supplementary Data 16, 17). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | ETS family members, particularly ETS1, regulate differentiation of keratinocytes and other epithelial cell types. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | ETS2 can function as a transcriptional repressor. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Anti-ETS2 and anti-H3K27Ac antibodies yielded higher ChIP-qPCR signals in iOE cells at the IRF6 -22 kb SNP than at an intergenic region (Fig. 5b, c), indicating ETS2 binds at this SNP and the SNP is within an active regulatory element. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | PCR and sequencing of chromatin precipitated by anti-ETS2 from iOE cells heterozygous for this SNP revealed an enrichment of ETS2 binding to the risk allele, while anti-H3K27Ac pulled down more of the non-risk allele (Fig. 5d, two additional replicates in Supplementary Fig. 17b). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | These findings are consistent with higher activity of the enhancer and higher expression of IRF6 in cells with the non-risk genotype. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Similarly, anti-ETS2 ChIP-qPCR in GMSM-K homozygous for either the risk or non-risk allele of IRF6 -22 kb SNP revealed stronger binding of ETS2 to the risk allele (Supplementary Fig. 18f). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | We found that reporter activity of the IRF6 -22 kb enhancer harboring the risk allele of the IRF6 -22 kb SNP was higher in ETS2-depleted HEKn relative to those transfected with a control siRNA (Fig. 5e and Supplementary Fig. 18g); we observed analogous results in GMSM-K (Supplementary Fig. 18h, i). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | IRF6 expression was also higher in ETS2-depleted HEKn or GMSM-K than in control transfected cells (Fig. 5f and Supplementary Fig. 18j). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Together these results support the model that the risk allele of the IRF6 -22 kb SNP results in lower expression of IRF6 by promoting the binding of ETS2, potentially among effects on the binding of other transcription factors (Fig. 5g and Supplementary Data 16, 17).Fig. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | 5Risk allele of IRF6 −22 kb SNP (rs661849) promotes binding of transcriptional repressor ETS2.a Consensus ETS2 binding motif from the JASPAR database of transcription factor DNA-binding preferences (Matrix ID: MA1484.1) and alignment of the variant site in several mammals. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele (C) is the reference allele but has a lower frequency than the non-risk allele (T) in most populations. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | The risk allele improves the match to the ETS2 binding site which remains partial. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | b, c Percent input identified by ChIP-qPCR for anti-ETS2 and anti-H3K27Ac respectively in iOE cells heterozygous for rs661849 using primers specific to the IRF6 −22 kb enhancer site or, as a negative control, to a region 103.7 kb upstream IRF6 transcription start site lacking ATAC-Seq and H3K27Ac ChIP-Seq signals in HIOEC or NHEK. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Error bars refer to three ChIP replicates and expressed as mean ± SD. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Each dot represents three technical qPCR replicates. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | NS non-significant. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | d Sequencing of anti-ETS2 and anti-H3K27Ac ChIP-PCR product of cells heterozygous for rs661849 using the indicated antibody. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | e Scattered dot plot of relative luciferase activity of the IRF6 −22 kb reporter construct (longest version) harboring the risk allele of rs661849 in control versus ETS2-depleted primary neonatal keratinocytes (HEKn). |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Data are represented as mean ± SD from three independent experiments. |
PMC12267437 | Identification of functional non-coding variants associated with orofacial cleft | Statistical significance (P value, two-tailed) is determined by Student’s t-test and P value is indicated on the plot. |
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