(A and B) ATAC-Seq profiles in control-derived inner ear organoids at day 8 (A) and day 13 (B) showcase dynamic open chromatin regions downstream of the GDF6 locus (chr8: ~95.72–95.75 Mb, hg38), highlighted with dashed ovals. ATAC-Seq data are from 1 representative biological replicate at day 8 and day 13. The tracks are shown to illustrate relative accessibility patterns at 2 time points as indicated. The orange arrow indicates the previously identified region in scATAC-Seq in 2D otic progenitor model as shown in Supplemental Figure 7. Black bars under the peaks indicate the statistically significant peaks called in ATAC-Seq. (C) Normalized ChIP-Seq densities (CTCF, H3K27ac, and H3K4me1) across approximately 250 kb downstream of GDF6 (chr8: 95,214,667–96,196,827) in control (CD-2) and patient (166-101) day 8 IEOs. The control shows strong enhancer signatures and CTCF binding at the candidate locus, which are absent in patient samples. The patient-derived deleted genomic region containing CTCF binding sites and CFAP418-AS1 lncRNA is indicated with a red line; the GDF6 locus is shown with dashed lines. The region shown was selected based on the pathogenic human deletions downstream of GDF6 and is not derived from genome-wide peak selection. The absence of enhancer-associated histone marks and CTCF signal in patient tracks compared with WT reflects deletion of the underlying genomic region shown with a red line. (D and E) Normalized ChIP-Seq densities for CTCF, H3K27ac, and H3K4me1, as well as ATAC-Seq densities around CTCF binding sites/candidate enhancers in the patient-derived deleted genomic region (D) and at the GDF6 locus (E) in patient IEOs compared with control at day 8. ATAC-Seq peaks overlapping with enhancer-associated marks and CTCF binding are shown with colored dashed lines in green and orange, respectively. CTCF binding sites are illustrated with red arrows. ChIP-Seq data are from 1 representative of 2 biological replicates for the control (CD-2) and 1 biological replicate for patient 166-101.