Journal: medRxiv

Article Title: A non-coding variant at 2p24.2 confers susceptibility to non-syndromic cleft lip and palate through LLPS-dependent regulation of MYCN

doi: 10.64898/2026.04.07.26350283

Figure Lengend Snippet: (A and B) FOXP2 overexpression upregulates MYCN . (A) Quantification of mRNA (RT-qPCR) and (B) protein levels (western blot) for FOXP2 and MYCN expression in cNCCs transfected with a FOXP2 overexpression vector (OE-FOXP2) or an empty vector control (OE-Mock). Data normalized to GAPDH . Mean ± SD ( n = 3). Unpaired two-tailed Student’s t-test. (C) Predicted FOXP2-binding motif. Schematic representation of the predicted FOXP2-binding sequence including the rs4263114 SNP within Enh- MYCN . (D and E) FOXP2 preferentially binds the non-risk allele at rs4263114. (D) Anti-FOXP2 chromatin immunoprecipitation (ChIP)-qPCR assay validating FOXP2 enrichment at the Enh- MYCN locus in heterozygous (T/G) cNCCs. Normal IgG served as the negative control. Mean ± SD ( n = 3). Two-way ANOVA (Sidak’s post-hoc test). (F) The risk allele abolishes FOXP2-mediated transactivation of Enh- MYCN . Relative luciferase activity of Enh- MYCN reporter constructs harboring either the non-risk (T) or risk (G) allele in HEPM cells co-transfected with OE- FOXP2 or OE-Mock. Mean ± SD (n = 3). One-way ANOVA (Tukey’s post-hoc test).

Article Snippet: For immunoprecipitation, the sheared chromatin was incubated with 2 μg of either anti-FOXP2 antibody (Novus Biologicals, NB100-55411, CO, USA) or normal rabbit IgG (Cell Signaling Technology, 2729S).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test, Binding Assay, Sequencing, Chromatin Immunoprecipitation, ChIP-qPCR, Negative Control, Luciferase, Activity Assay, Construct

Journal: medRxiv

Article Title: A non-coding variant at 2p24.2 confers susceptibility to non-syndromic cleft lip and palate through LLPS-dependent regulation of MYCN

doi: 10.64898/2026.04.07.26350283

Figure Lengend Snippet: (A and B) FOXP2 contains a prominent intrinsically disordered region (IDR). Bioinformatic prediction of the FOXP2 IDR sequence using (A) PONDR and (B) PLAAC, providing the structural basis for phase separation. (C) The risk allele disrupts the formation of FOXP2 nuclear condensates. Representative immunofluorescence images of FOXP2 in cNCCs harboring heterozygous (T/G) or homozygous risk (G/G) alleles at rs4263114. Droplet-like FOXP2 condensates are prominent in T/G cells but show a diffuse distribution in G/G cells. Scale bars, 20 μm (left) and 5 μm (right). (D and E) FOXP2 condensates show highly dynamic liquid-like properties. (D) Representative time-lapse fluorescence recovery after photobleaching (FRAP) images of EGFP-FOXP2 overexpressing HEPM cells. Scale bar, 5 μm. (E) Quantification of normalized fluorescence recovery intensity over time ( n = 3 independent experiments). (F) LLPS is required for the allele-specific regulatory activity of Enh- MYCN . Dual-luciferase reporter assay comparing the activity of Enh- MYCN constructs harboring the non-risk (T) or risk (G) allele in HEPM cells upon treatment with 1.5% 1,6-hexanediol (1,6-HD) to chemically disrupt LLPS. Mean ± SD ( n = 3). Unpaired two-tailed Student’s t-test. (G-I) Overexpression of the FOXP2-IDR domain rescues phase separation and MYCN expression in risk-allele cNCCs. (G) Immunofluorescence staining of FOXP2 showing restoration of nuclear condensates in homozygous risk (G/G) cNCCs after FOXP2-IDR overexpression (OE-IDR) versus a Mock-vector baseline (OE-Mock). Scale bar, 10 μm. (H) RT-qPCR and (I) western blot analyses of FOXP2 and MYCN mRNA levels, and their corresponding protein levels, in OE-Mock and OE-IDR G/G cNCCs. Data normalized to GAPDH . For (H) Mean ± SD ( n = 3). Two-way ANOVA (Sidak’s post-hoc test).

Article Snippet: For immunoprecipitation, the sheared chromatin was incubated with 2 μg of either anti-FOXP2 antibody (Novus Biologicals, NB100-55411, CO, USA) or normal rabbit IgG (Cell Signaling Technology, 2729S).

Techniques: Sequencing, Immunofluorescence, Fluorescence, Activity Assay, Luciferase, Reporter Assay, Construct, Two Tailed Test, Over Expression, Expressing, Staining, Plasmid Preparation, Quantitative RT-PCR, Western Blot