Supplementary Material for - Nature Research€¦ · Supplementary Material for Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease. Noor

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Supplementary Material for Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing

NCRNA disease.

Noor M. Ghiasvand, Dellaney D. Rudolph, Mohammad Mashayekhi, Joseph A. Brzezinski IV, Daniel Goldman and Tom Glaser

Supplementary Figures 2

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Supplementary Note 24

Supplementary References 26

Supplementary Tables 33

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Suppl. Table 3 36

Nature Neuroscience: doi:10.1038/nn.2798Nature Neuroscience: doi:10.1038/nn.2798

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Supplementary Figure 1. Map of the 10 kb genomic region surrounding ATOH7, showing the PCR amplicons screened for mutations (yellow boxes, described in Suppl. Table 1). The major 1.7 kb transcript (green), coding sequence (CDS, red)1 and AluSb insertion-deletion polymorphism2 are indicated. The 0.35 kb AluSb element was present in the NCRNA haplotype and 13 of 15 chromosomes tested, but is absent from the NCBI reference genome. ATOH7 contains a single exon and is not spliced in the retina3.


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Supplementary Figure 2. Confirmation of deletion endpoints. A. Map showing nested junctional PCRs. B. Products were amplified from NCRNA homozygotes (rr) but not from wild-type (RR) DNA samples. C. Flanking PCR primer pairs. The sequences are given in Suppl. Table 3, except primers 29.5 FOR (5’- AGGAACAAGGATACCTTCACT) and 37.7 REV (5’- CTGTTTGA-AATTCTCCATTACA).


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Supplementary Figure 3. High resolution SNP (single nucleotide polymorphism) analysis of the NCRNA region.


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Supplementary Figure 3. High resolution SNP (single nucleotide polymorphism) analysis of the NCRNA region. Six DNA samples from the Khorasani pedigree were evaluated for 1.1 million biallelic SNPs using Illumina Omni1-quad BeadChips. The markers are spaced 2.4 kb apart on average, genome-wide. This diagram shows data for 390 SNPs from the 1.6 Mb critical region, analyzed using GenomeStudio software. A. Genotype plot (B allele frequency, BAF). Each row represents a different DNA

sample. Values close to 0.5 indicate heterozygosity. B. Map of the 10q21 critical region4, bounded by D10S1670 and D10S1418 (vertical blue

lines), showing candidate genes and the NCRNA deletion (vertical yellow box) in a genome browser window (Build 36.1 hg18).

C. Signal intensity plot (log R ratio) showing the normalized dosage of each SNP marker.

Values close to 0.0 indicate two (diploid) copies. The top two rows in panels A and C show blood (10859) and EBV lymphoblastoid (10860) DNA from the same blind individual (rr) who is homozygous across the entire region. The bottom rows show archival samples from two carriers (10883, 10885 Rr) and a normal sibling control (10886 RR), who are heterozygous for roughly half of the SNPs; and a blind individual (10877 rr) who was not tested in previous studies. Sample 10877 (rr) is heterozygous for SNPs centromeric to MYPN, and homozygous for the telomeric SNPs, indicating a novel recombinant disease haplotype. The underlying crossover (vertical red line) divides the critical region in half – narrowing the nonrecombinant (NR) interval from 1639 kb to 713 kb – and formally excludes four candidate genes. These include SIRT1, which has a severe retinal dysgenesis phenotype in mutant mice5. SNP rs3858145 (circled) is deleted in all three blind samples (R << 0.1) – so the corresponding BAF values are not meaningful (nc, no call) – and it is hemizygous in both carriers. No other SNP in the 1.6 Mb region exhibits this pattern. We conclude there are no further deletions (or duplications) associated with NCRNA disease in the 10q21 interval, at this level of resolution. D. Polar θ ×R plot of rs3858145

genotypes, created using GenomeStudio software.

In this plot, norm R values close to 1.0 indicate two (diploid) copies. The wild type sample (10886 RR) is inside the blue ellipse, and the two hemizygous samples (10883; 10885 Rr) are below. The blind individuals (10859, 10860; 10877 rr) have no signal (baseline), so their norm Theta (BAF) values are not meaningful (no call).

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Supplementary Figure 4. RT-PCR analysis of genes flanking the NCRNA deletion, performed using mouse tissues as described in the Materials and Methods. Atoh7 is specifically expressed in the developing eye, whereas Mypn and Pbld are mainly transcribed in the adult heart and kidney, respectively. Consequently, human MYPN loss-of-function mutations cause familial cardiac myopathy, with no ocular pathology6. Apart from Atoh7, no gene in the nonrecombinant interval has a mouse tissue expression profile (Unigene) matching the NCRNA disease pathology (not shown). The PCR primer sequences are: GENE forward primer [5’-] reverse primer [5’-] Atoh7 (Math5) CGCCGCATGCAGGGGCTGAACACG GATTGAGTTTTCTCCCCTAAGACCC Pbld TTACGCCAGTGAGTGAAGTCCC TCAGCAATACCAACCCACGG Mypn TGGGACAGATTCAACTTCCGC CCGAAAACACCTCAGCAATGAC Actb (β-actin) GCTCTTTTCCAGCCTTCCTT GTACTTGCGCTCAGGAGGAG


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Supplementary Figure 5. Comparison of transgene expression in wholemount E14.5 albino littermates carrying the 3034-BGnCherry (left) or Math5-GFP (right) reporter.


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Supplementary Figure 6. Phylogenetic analysis of the ATOH7 region. Orthologous flanking genes were identified for eleven assembled vertebrate genomes in the NCBI database. Arrows indicate curated genes and the direction of transcription. The gene order is conserved among tetrapods. There is an apparent inversion of the Anolis HNRPH3-PBLD segment (bracket). A gap in Xenopus tropicalis scaffold_244 includes PBLD, but this gene (light green arrow) is represented as a curated cDNA (accession BC155870). Sequences with similarity to the human remote enhancer were identified with confidence in all tetrapod genomes, in the interval between PBLD and ATOH7 (small gray circles). In teleosts (yellow box), this syntenic block has been disrupted by multiple evolutionary translocations, inversions and insertions, with breakpoints close to the ATOH7 promoter in all species examined. These exclude the existence of an orthologous, physically remote regulatory unit. However, short stretches of similarity (broken circles) were detected in some teleosts within 2.5 kb of the start codon (Suppl. Figures 7c and 8c). In the zebrafish genome, ATOH7 (ath5) is separated from PBLD and MYPN by 2.7 and 23.4 Mb, respectively, on chromosome 13. Genome builds: Homo sapiens human (GRCh37/hg19), Mus musculus mouse (NCBI37/mm9), Monodelphis domestica opossum (Broad/monDom5), Gallus gallus chicken (WUGSC 2.1/galGal3), Taeniopygia guttata finch (WUGSC 3.2.4/taeGut1), Anolis carolensis lizard (Broad/anoCar1), Xenopus tropicalis frog (JGI 4.1/xenTro2), Oryzias latipes medaka (NIG/UT MEDAKA1/oryLat2), Gasterosteus aculeatus stickleback (Broad/gasAcu1), Danio rerio zebrafish (Zv8/danRer6), Tetraodon nigroviridis pufferfish (Genoscope 8.0/tetNig2) and Petromyzon marinus lamprey (WUGSC 3.0/petMar1).


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Supplementary Figure 7. Intergenic conservation of the 5’ ATOH7 regulatory region.


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Supplementary Figure 7. Intergenic conservation of the 5’ ATOH7 regulatory region. A. VISTA plot showing the percent identity between a 53.6 kb human DNA segment and five vertebrate genomes aligned using mLAGAN, for a sliding 50-nucleotide window. Orthologous CDS and CNEs with >70% identity are shaded different colors. Reporter transgenes used in our study are indicated at the top (red and green bars), along with the NCRNA deletion and interspersed human repeats. The primary enhancer has a bipartite structure, with two distinct functional units7,8. The proximal subregion (blue peak) is sufficient to confer autogenous neuroretinal expression in Xenopus7,9, chick10,11 and zebrafish12,13 embryos, and this activity depends on the presence of specific E-box bHLH binding sites (Suppl. Figure 8e). The distal subregion (green peak) is sufficient for retinal transgene expression in Xenopus and mice, and its main activity resides in a 340 bp core that is 1.5 kb upstream from the mouse Atoh7 promoter7,8. The shadow enhancer contains three separate CNEs. Weak homology was detected for CNE3 (yellow peak) between human and stickleback DNA (arrow). A further upstream element at -38 kb (CNE4) has unknown function but is highly conserved among tetrapods. B. Distribution of consensus binding sites for bicoid (K50, red) and paired (Q50, blue) homeodomain (HD), ETS (Pea3 and ERM), and bHLH (E-box) transcription factors. The Q50 sites (YTAATTAR) recognized by paired-type HD proteins (e.g. Pax6, Chx10, Rx) are clustered in the CNEs (see legend in Suppl. Figure 7e). C. Evolutionary origin of the shadow enhancer. Diagram showing the physical position of CNEs in different vertebrate species. The shadow and primary enhancers are ~20 kb apart in human DNA, whereas in the trace homology to CNE3 (yellow) in the stickleback genome is located 2 kb upstream from the start codon (Suppl. Figure 8c). The dual organization of ATOH7 enhancers thus appears to have evolved from a single cluster of regulatory elements in the common ancestor of bony vertebrates, through accumulation of intervening repeats, which has progressively increased the physical separation between CNE modules over evolutionary time. The dispersion of CNEs scales roughly with genome size and may be correlated with increasing regulatory complexity14. Inset. The distance separating Atoh7 from its nearest upstream flanking gene is plotted against genome size for the seven species analyzed. CDS, coding sequence; CNE, conserved noncoding element.


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Supplementary Figure 7 (continued). Intergenic conservation of the 5’ ATOH7 regulatory region. D,E. Enlarged view of shadow and primary enhancers showing consensus binding motifs in human DNA. Core K50 and Q50 homeodomain sites have been defined experimentally15-18. The ETS factors Pea3 and ERM mediate FGF signaling19-23. E-boxes required for Xenopus, chicken or zebrafish transgene expression7,10,13 are outlined in red.


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Supplementary Figure 8. Multi-species alignments for five core CNEs, generated by mLAGAN and pairwise BLAST. Sequences are numbered from the ATOH7 start codon (ATG) and correspond to CNEs in Suppl. Figure 7a. Matches to the human genome are shown in color. A-C. Alignments from the shadow enhancer. The principal genotyped SNP (rs3858145) associated with variation in optic disc area24-26 among healthy individuals is indicated in CNE2 (panel B). This SNP is located in the core of a conserved E-box (CAAT/CTG), which is the consensus DNA binding site for bHLH proteins (CANNTG). Haplotypes containing the C allele (as shown, or G in the NCBI reference ‘top’ strand) are correlated with a reduced number of RGCs. D,E. Alignments from the primary enhancer. The TATAAA motif, predicted transcription initiation site (for human), and conserved E-boxes with major transcriptional effects7,10,13 are indicated. Two of the human-teleost alignments were statistically significant in the pairwise BLAST analysis, using seed word size = 7 and default parameters. Human CNE3 (535 nt) and proximal CNE (429 nt) queries were aligned versus a 52 kb segment of stickleback DNA spanning Atoh7, with 59/83 (72%) and 40/56 (72%) identities, and expect probabilities P = 5e-04 and P = 0.018, respectively. Sequences with similarity to human shadow (CNE3) and primary (distal CNE) enhancers appear to be contiguous in teleosts (yellow boxes in panels C and D). No similarity was detected between any of these sequences and the 9.6 kb segment upstream of the sea lamprey Atoh7 gene (Petromyzon marinus Contig9055/PMAR3). F. Alignment from CNE4.


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Supplementary Figure 8A. ATOH7 remote enhancer (CNE1) human -22057 CTAAAAATCACGATTTCAGCATAAAAGAAAAGAAGA--GGT -22019 mouse -15065 CT-AAAGTCATGATTTCAGCATAAAAGAAAAGAAAG--GCT -15028 opossum -24361 CCCGAAAACAGCACTTCAGCAGAAAAGAAAAGAAGGGAGCC -24312 chicken -11125 ------GTTACAATTTCAGCAGAAAAAAAAAAAA------A -11097 lizard -11613 ------GTAACAATTTCAGCAGGAAGAAAGGAAC------- -11586 Xenopus -10673 -----CGTTAGAATCT------------------------- -10663 human -22018 AAGTTAAAAGTGAACACCTGTTCTTCTAAAATTGCTCTACTGCA------ -21975 mouse -15027 AAGTTAAAAGCCAGCACCTGTTCTTCTAAAATTGCTTTTCTGCA------ -14984 opossum -24311 GGGGTAAAAGTGAACACCTGTTCTCCTAAAACTGCTCTATTGCA------ -24268 chicken -11096 AGATTAAAAGGAAACACCTGCTCCT-GTAAATTACTCTGCTGCA------ -11054 lizard -11585 --ATTCAAAAAGACCACCTGCTCTTAGAAAATCACTCTGCTGCT------ -11544 Xenopus -10662 --GTTA---------TCCTG------TACATTTG-GCTGCAGTACGCTAT -10631 human -21974 ----GATACCAGCCCTAGAGAGTTGGCAGATGGTTTTGTGTTTCTAAGCC -21929 mouse -14983 ----GGTAGCAGCCCCAGAGAGCCAGCAGATGGTTTTGTGTTCCGAAGCC -14938 opossum -24267 ----GGTATCAGCCCAGGAGAGTGTACAGATGGTTCTGTGTTTGCAAATC -24222 chicken -11053 ----GGTACCAACCCAGGAGACTCTGCAGATGGTTCTGTGTTTGCAAACC -11008 lizard -11543 ----GGTCCTGGCCCGGGAGAGTTTGCAGATGGTTTTGGGTTTGCAAGCG -11498 Xenopus -10630 GTGTGGGGCCAGCGCGCG-------GCAG-------TGTG---------- -10605 human -21928 TGACCTTAGCAGCCCTTCATCAATCTTGAGCTGACTGATTCACAGGAAAG -21879 mouse -14937 TGACCTCAGCAGCCCTTCATCAATCTTGAGCTGGTTGACTC--------- -14897 opossum -24221 TGACCTTCGGAGCCCTTCATCAATCTTGGGCTGATTGATTCTCAGGAAAG -24172 chicken -11007 TGACCTCAGCACCCCTTCATCAATCTCTGGCTGATTGATTCTTTCCAAAG -10958 lizard -11497 TGACCTCAGCAGCCCGTCATCAATCTTCAGCCGAGTGATGCTTCTGAAGC -11448 Xenopus -10604 ----------ACTGCTTCCTTAAT-------TGATTGG------------ -10584 human -21878 AGGAGACTCCAGCTTCCCAGGGTACC-CAAGACATCTTTGATTTAGTTAT -21830 mouse -14896 -----TCTCGGGCTTCCCAGGGTACC-CAAGATATCTTTGATGTACTTAC -14853 opossum -24171 CCGAGTGGCT-GCTTCCCGGGGCGCCGCAAGACACCTTCGAGTTAGTTAC -24123 chicken -10957 TGGAGCACGCCATGTCCCGGGGTGCTGGAAGACACCTTTGCGTTAGTCCC -10908 lizard -11447 GGTGGACGTTCTGTTCC------------------------------CCT -11428 Xenopus -10583 ------------CTTT----GTTGTG-GTTGAGTCGGCGGCGTTAGTGTG -10551 stickleback -3004 ----------TGCGTCCGTGG----------------------------- -2994 human -21829 TACCTGAAGACCAGTTAATTCAGCCACA-----CTGATTCTCTCAAGTTT -21785 mouse -14852 TACCTGAAGACCAATTAATTAGCCCATA-----GCGATTCTGTTATGGTT -14808 opossum -24122 TACCTGAAGACCAGTTAATTGACGCACG-----CTGCTTCTTTCAGGCTT -24078 chicken -10907 GGTCGGAATACCAGCTAATTGGTGCTTT---TTTTCATTTTTGGAGGTTC -10861 lizard -11427 GTCGGGAAGGCTAGTTAATTTGCGCTTTGGCTTTTTGCTTTTCTTGGTTC -11378 Xenopus -10550 GTGCGGA-------------------------------TCCGGGAGGTTC -10532 stickleback -2993 ---------------TAATTTACACCCT-----TTGGTT----------- -2975 human -21784 TCC -21782 mouse -14807 TCC -14805 opossum -24077 TTC -24075 chicken -10860 TTA -10838 lizard -11377 TTC -11375 Xenopus -10531 CCA -10529


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Supplementary Figure 8B. ATOH7 remote enhancer (CNE2) human -20736 AGTGTCACATTCTATTATACTTGATGGG-GAAACTC-GAGGCGTCCTGGT -20689 mouse -10778 AATGTCACATTCTATTTTGCTTGATGGGAGAAATTC-AAGGCGTCCTGGT -10730 opossum -20416 AGTGTCTCATCTTATTACCCT-GATGGGA-AAA-TGGGCGGCGTCCTGGT -20370 chicken -8836 AGGACTGCATTTCTTTTT----GATGGAAAAACCTCTGTGGCGTCCTGCT -8791 lizard -8739 TGGACTATATTTGACCTTTCCTGAAAGGTCTACTTCTGAACTTTATTCTT -8691 Xenopus -9346 AAGACATGA------GTAGGTAGAAAAGGCACCTGCCTAGCACCCCCCCC -9304 human -20688 GGCAGGCA-----------------------------GGACAAAAGGACA -20668 mouse -10729 GGCTAGCA-----------------------------GGACAAAAGCCCA -10709 opossum -20369 GTCCAACA-----------------------------GGACAAAAGAGCA -20349 chicken -8790 GTCCATTT-----------------------------GGACAAAAGAGCA -8770 lizard -8690 CACATGTTTTTATTGACTTTATATATTCCTTTAATAAAGATATTAGAT-A -8641 Xenopus -9303 CCCAT--------------------------------------------A -9297 human -20667 GCTGTGCTGGGCTGCCCACAAATTAGGGCATCTTTGGAGGCTGTCCTCTG -20618 mouse -10708 GCTGTGCCGGCCTGCACACAAACTAGGGCATCTCTGGAGACTACCTTCCA -10659 opossum -20348 GCTGGGA-GCAGAGAGTGAGTGCATGGATGTGTGTGGGGTGAGGCAGGGG -20300 chicken -8769 GATGTGT-------------TATATGCGCGAATGCGTGTGTGCTCATCTG -8733 lizard -8640 GATT--CTGG----------TCTCTGCGCATGGTTATTGGTGCTCTGCAG -8603 Xenopus -9296 TTTGCAC-------------CATAGGC---------------CTCTTCTG -9275 human -20617 ATTCTCAATTAATTTTTAGGAGAGCTTTAATTACGCCACCAACAGTCCTC -20568 mouse -10658 ATTTGCCCTCAAATTCTGGGAGAGTTTTAATCATGTCACCAACCAGCCTC -10609 opossum -20299 CTTCAGGATTCATTTCCAGCAAAGCTTTAATTACTGGGGCTCCAGCAGTC -20250 chicken -8732 CCTGTGGCAGGATACAAAAAATAGAGCTA--------------------- -8704 lizard -8602 CCTGGGTCTTGACATACATTGAGGC------------------------- -8578 Xenopus -9274 CCTAC--------------------------------------------- -9270 human -20567 ACT------------TGAACAATCACAAGCACTTCAGCTTGGGGGGAAAA -20530 mouse -10608 GCTGCTCTGATCTCAGGAACAATTGTAGGCACT--AGGCTGAGTTGGGAA -10561 opossum -20249 ACCC---------------AAAGCAGGAGCTGCACAGGAAGATCTGTGTG -20215 chicken -------------------------------------------------- lizard -------------------------------------------------- Xenopus -------------------------------------------------- human -20529 GAGCGACTGACTGGATTTGCATAATCATTTGCCCTAATTAGAATGTCAAA -20480 mouse -10560 AAGTGACAACCCTGATTTGCATAAGCATGTATCCTAATTAGAATGTCAAA -10511 opossum -20214 GAATGCCTGC----ATTTGCATAACCATTTAGCTTAATTAGAATGTCAAG -20169 chicken -8703 -----CCAAAGTTCCTTGTTAAGTTGGAAAGCATTAATTACAACATCAAG -8659 lizard -8577 --------TGGGTGGCCATCTGTCAGGGATGCTTTGCTTGCGCTTTTGTG -8536 Xenopus -9269 -----CCGTAGTTCC----------------------------------- -9260 human -20479 AGTTGGTGGCAAAGAGGCCTAGTTGTGCT-GTGACTTAGGATCATTTTAA -20431 mouse -10510 AGTTGGCGGCAGAGAGGTGGTGTTCTACTCTTGACTTTTGATCATTTTAA -10461 opossum -20168 AGTTGGTGCCAGAGTTGTGAAAA--------TGACTTAGTATCATTTTAA -20127 chicken -8658 CCTTACAGTTAACAGTGTA------------------------------- -8640 lizard -8535 CACAAAGGCAGAAGGGG----GTTGG------------------------ -8514 Xenopus --------------------------------------------------

rs3858145 human -20430 TTTAGTCCAATTTCCCATTATTACAATTGCCTTCCTCTTTGCACCT--CA -20383 mouse -10460 TTTCCTTCAATTTCCAGTCATTACAACTGTCTCC-------TAACT--CA -10420 opossum -20126 TTTAGCCCAGTGTCCCATGGTTAACTTTCCTCTTCTTCCTTTCCCTCCCA -20077 chicken -------------------------------------------------- lizard -8513 ----ACTAAATGGCCCAAGGGGTCTCTTCC-------------------- -8488 Xenopus -------------------------------------------------- human -20382 GATCCTCTTTC -20372 mouse -10419 GGCCCTCCTTT -10408 opossum -20076 AACCCTCCTCT -20066 chicken -8639 AACCTTTTTTT -8629 lizard -8487 AACCCTCTTTT -8477 Xenopus -9259 AGCCCTGCCTT -9249


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Supplementary Figure 8C. ATOH7 remote enhancer (CNE3) human -19907 TCTCAGGCATAGAACCACCCTGACCCATTAG-AGACTGCTTAGGCATGCA -19859 mouse -10043 TCTCAGGCTTAGAACCGCCCCGACCCATTAGCAGGGGTCTTAAGTATGCA -9994 opossum -18261 TCTCCTGCACAG-TCCACTCAGGCCCATTAG-AAGCTGTTAAGGTATGCA -18214 chicken -7526 TCTCATTTATTTAACAGCTTTGACCCATTAG-GGGCTTTTAAATCATGCA -7478 lizard -7122 TCTCATTTATGTAACTCATATAGTCCATTAA-AGGCCTTCAGAACATGCA -7074 Xenopus -8340 ACATTTTTTTTCTACCCCTTTTTTCAATATAATAGCTT-----------A -8302 human -19858 TCGGTGATTTCTGGGTCTGGCTGGTAAGCTAC-----------------T -19826 mouse -9993 TCGGTGATCTCCGAGCCTGCTTTTTGGGC--------------------- -9965 opossum -18213 TCAGTGGT-TGGGGCTATCTCTGAGAAGCTCC-----------------T -18182 chicken -7477 TCACTGACTTC-AGGCTTGCTC----TTGT-----CAAG--------CTA -7446 lizard -7073 TTACTGACCTCTAGGATTG--------TCTTTGTCTAACTGCT-----TG -7037 Xenopus -8301 TCTTTGCCTTC-A--TTTGCTCAGTATTGTGTGCCCAAGTGTTTTATCTA -8255 human -19825 TACCCAGGTCCTAGTTAAATCAGGCATCCTCTG----------TTTTTCC -19786 mouse -9964 --------TCCCAGATTAATCAGGCATCCTC------------TCCTTCC -9935 opossum -18181 GACTCTCACCCTAGTTCAATCAAGCATCCTCTCTCCCCATCCCCATCCCC -18132 chicken -7445 CTTAAAGATACATGTTAAATCAGGCATTTCATAT---------TTTTGC- -7406 lizard -7036 AATAGCGATCTCAAGGAAATGGGTAATTTTCTAATGTTT----TCTTGCC -6991 Xenopus -8254 AATAAAACTGTATGTAAAATATGACATATACTACCCGCTTA--TCCTGCC -8207 human -19785 GCCCTCATTCCCTTTTA--------ACAGCAGGA-----GGCCACCTATT -19749 mouse -9934 GCCCTCATTCCTTTCTGCGGT----GGGGGTGGG-----GGCCATCTTGT -9894 opossum -18131 ATCCCCCTT-----TTAT--------CAGCAGGA-----GGCCACATCCT -18095 chicken -7405 --CCTCATTCGCTTTTGTT----TACCAGCAGGA-----GACAAAATATT -7367 lizard -6990 TTCCTCTTT-----TTAGGT--TGATCAGCAGGA-----GGCAAAATATT -6953 Xenopus -8206 GCCTCATTAGGTGGCTCCTGTCATTCTGGAAGCACTGCTGATTAAATATT -8157 human -19748 TAATTAGTTG-GGGGTGGGAGCGTCTGTAATCTCCTGGGGGTGG---CTT -19703 mouse -9893 TAATTAGAT----GGTGGGAGCGTCTCTAATCTCTTGGGGGTGG---ATT -9851 opossum -18094 TAATTAGTTGGGGGTGGGGACCACTTGTAATCTCCTGGGAGCGGCCAATG -18045 chicken -7366 TAATTAGTTTTGGGGCA------TTTATAATCTCCCCTGGGCAA---GCT -7326 lizard -6952 TAATTAGATTGGGGGCGTC----CCTGATCTCTCTCCTGGGCCA---GCT -6910 Xenopus -8156 T-ATTTGTAATGATCCAT-----TTTATTGTCTTCC-------A---GC- -8124 human -19702 CA-GCCCCAGGGTTGGATAAGAGGTTGAATCCAACGCCAGGTG--TGGCT -19656 mouse -9850 TAGGCTCCAGAACTAGATTAGAGGGTGATTCCAGCACCAGGGGCATGGCT -9801 opossum -18044 GCCTGGCTGGTGCTAGACGCTTGGTTGGAGTTAATGCCAGGCTT--GGTT -17997 chicken -7325 CCTGCGACAATGCTGGACGCCACTTCTGAGTTAATTCCTGTCATG---CT -7280 lizard -6909 CCAGCCATCCTGCTGGACGCCACTTTGGGGTTAATTCCAGTCAG------ -6866 Xenopus -8123 ---------GTGATG-ATA--------AAATTAGTTACAGTAATAGGATG -8092 human -19655 GGCC-TCTCC-CCTGC------------AACATATGGGGAACCCACCACA -19619 mouse -9800 GGCC-TCTAT-CCTGC------------AACATATGGGAAACCTGCCAGA -9764 opossum -17996 GGCC-TCTCCGCCCAT------------GACATATGGGAAATCCTTCGAG -17959 chicken -7279 GGCC-CCT---CCAGTAC----------AACATATGGGTAGCCTATAAG- -7243 lizard -6865 -----------TCTGGGCACAGAACGAGAACATATGGGGAGCCCTGGA-- -6829 Xenopus -8091 TGATTTCT---TCTGTCC----------ATTA-ATGGAT----------- -8067 human -19618 TTTTCAGCCCAAGTTC------CAGTAGCAAGCATTAGTGGAG-AGGCCT -19577 mouse -9763 CCTTCAATTGGAGTTC------CAGCAGCAAGCATAAGTGAAG-GAGCCT -9722 opossum -17958 CTTCCATCCAAGGTTA------CAACAGCCAGTTCTGGGGGAGTGGAGCT -17916 chicken -7242 CCTTCAGCAAAAGTTGC------AACTG-AACTATTAGAGGAGTGGGACC -7201 lizard -6828 --------------------------AGACCTTTGTACAGGCCGAGAGCC -6805 Xenopus -8066 ---------TAAGTTACTCATTTAATAAAACGC-TTGGCCAAACATGAAA -8027 human -19576 TCCCTGCTGGGAACACACCCCGCAAAGC-------CAAGGGCAGGCGGCG -19534 mouse -9721 TCCCTGCTGGGAACGCACCCTGCAAAGC-------CAAGGGTGGACAGCA -9679 opossum -17915 TCCCTGCTGGGAAAATAACCTGCCACAAAGGACTGAGAGGACCAGCGTCA -17866 chicken -7200 TCCCTGCTGGGAAAATACTGGGCAACAAAGCA---AAAAGGGGCCA-GCG -7155 lizard -6804 CCCCTGCTGAGGAAACACTGGGCAACAAAGCC---AAAAGGCAGCCGACG -6758 Xenopus -8026 TCTCTGGTAGGA----------CAAACATGTA---GAAAAGAATCAGTTC -7990


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human -19533 TCACTACTAGAGGTCAAGGCA----GTTCTT----AGACCAAACAGCATC -19492 mouse -9678 TGGCCAGGAGAGGCCAAGGCA----GTTCTT----AGACTAGGTAGCATC -9637 opossum -17865 TCCCTGGTAGAGGTCAAGGCA----ATACTT----AGGCCAAACAGCATC -17824 chicken -7154 TCACCGGTGGAGGTCAAGGCA----ACACT----CAGACCAAACAGCATC -7113 lizard -6757 TCACTAGTGGAAGTCAAAGCA----GCACTT----AGACCAAACAGCAGC -6716 Xenopus -7989 CCACCAGT--CAGATAAGACATGTACAGCTTTAGCAGCACAGACGACATA -7942 stickleback -2088 -----------------------------------AGGACAAGCAGCAGC -2074 medaka -1264 -----------------------------------ACGACCAGCAGCAGC -1250 zebrafish -2378 -----------------------------------GGGACAAGCAGCAGC -2364 human -19491 TGGCAAACTGTGCCAATTGGAAATCAATGGC-------CCTCCCTGGG-- -19451 mouse -9636 TGGCAAGCTATGCCAATTGTCAATCAATGGC-------CCTCCCTGGG-- -9596 opossum -17823 TGGCAAGCTGGGTTGATTGGAAATCAATGGG-------ACGCCGCAGGGC -17781 chicken -7112 TGGCAAACTGTGCCAAGTAGAAATCGATGAGAAG---------CAGGGCA -7072 lizard -6715 TGTCATGTCTTCTTCATTGGAAATCAATGGGAAATGAA-------GGGTT -6673 Xenopus -7941 TGGCAAGCAGTTCCAATTGGAAATCAATGAG-------CCG--CTGA--- -7904 stickleback -2073 TGGCAAACTACATCAATCTGTAATCAATGAA-------CCAA-CTGGGAG -2032 medaka -1249 TGGCAAACTACACCAGTCTGTAATAAATGAA-------GCGA-CTGGTAG -1208 zebrafish -2363 TGGTCAGGGATGCCAATCTGCAATCAATGAAAACAAACTAAACCAGTGGA -2302 human -19450 -TGGGTT-TAATTTTA-TGGTTATTTGCAC -19424 mouse -9595 -TGGGTT-TAATTTTA-TGGTTATTTGCAC -9569 opossum -17780 ATGGGTG-TAATTTTA-TGCCTCTCTGCAC -17753 chicken -7071 ATTGGTAGAAATTTTA-TGCCTATTTGC-- -7045 lizard -6672 GTAGGTTGGAATTTTA-TGCCCGCTTGCAC -6644 Xenopus -7903 ---AGTATAAAGGTTACCACCCCTTTGC-- -7879 stickleback -2031 ATGGG-----ATTTTA-TGCCTAATTGCAC -2008 medaka -1207 ATGGG-----ATTATC-TGCCCAAATGCAC -1184 zebrafish -2301 TTGGGT-----TTATA-TGCCCAATTGAAC -2290


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Supplementary Figure 8D. ATOH7 primary enhancer (distal CNE) human -1943 ATATTTGACATTTTGATGACTATATA -1918 mouse -1821 ACGTTTGACATCCTGATGACTAAAGA -1796 opossum -2284 GTGCATAATTTGGTAACCAAGTCTTG -2259 chicken -1943 TCGTACGTTTTGATAATGGAATAATG -1918 lizard -1972 TCAGATGTTTTGCTAATGAAATAATG -1947 Xenopus -4163 TGGCTGATTCTCATAAGG------TG -4144 human -1917 ACATAGTCTTTTCTGTCACAGGACTGCTGTTCCCAACCAAGACTGTTATG -1868 mouse -1795 ACACAGTTGTTCCCAGCGCAGAACTGCTGCTCCCAA-CGTGACTGTTAGG -1747 opossum -2258 TCATACTCCCATC-ATAGCAGGACTACCATACTC--------TCATCATA -2218 chicken -1917 GAGACTCCCTACCCCAACCATCATCTCTGTTCAAGACCA------TTAGG -1874 lizard -1946 ATGTCTTACCACCCAGCCATTAA--------CAAGACCAT-----TTAGG -1910 Xenopus -4143 GGTGCCTCC----------------------------------------- -4135 human -1867 ACCCA-AAGTAACAGAGCTGAGTGGCTCCATGC--AGAAGTCTGTGTTCA -1821 mouse -1746 ACCGAGGAGTGATGGAGCTGAGTGGCTCTGTGC-AAAAAGTCTGTGTCCA -1698 opossum -2217 GTGGCCAAGGCACAGGGAGGAGCGT-----------ACAGCCTGCATCCA -2179 chicken -1873 ATTCAGAATATTAAGAGCAGACCAACTGGGCGT--AACAGCCTACGTTCA -1826 lizard -1909 TTGCGGGATATTAAGAGCGGGGTGATTTTGCCT--AACAGACTGGGTTCA -1862 Xenopus -4134 --------------------ACCAGCTG-ATG---ACTGACCTCAATTCA -4109 stickleback -2007 ---------------AGCTAACCAG-TCCATCCCAAAATGAC-CAG---- -1976 medaka -1183 ---------------AGCTCACCGG-TCAATCT---GAAGAC-CAG-CAG -1155 zebrafish -2289 ---------------AGCAGAATTACT-CATCTAATGCAGA-TGAG-CGT -2258 human -1820 ACATCTGTCATGCTGGAGATCATTAATGTTCAATTGCTTCCTATTGTCTT -1771 mouse -1697 ACATCTGTCGCTCTGAAGATTATTAATGTTCAATTGCTTCCCATTGTCTT -1648 opossum -2178 ACATCTGTTAGACCAGAGTCCATTAATGTCCAATTGTATCCCCTTGTCTT -2129 chicken -1825 ACATCTGTCGTGCTCAAGTCCATTAGTGTTCAATTGCATCCTATTGTTTT -1776 lizard -1861 ACATCTGTTATGCTGGAGTCCCCTCATGTTCAATTGCGTCCCATTGTCTT -1812 Xenopus -4108 ACATCTGTCATGTTGGAGTCAATTATTTCACAAGTGCATCCTATTGTCCC -4059 stickleback -1975 ACAGCA-TCA-GCTTGTGTCCA----TGACCCAATACGAGAGAAGGG--- -1938 medaka -1154 ACAGCA-------TG-AGAC-------GGGCG--TGGGTCC--------- -1131 zebrafish -2257 ATATG------GCTTGA---------TGGCCACA---GTC--AGCCGGGA -2228 human -1770 CATGTCATTCAACTTCATTTCATTGTCATTGCTTCAGCTGGGGGATTAAT -1721 mouse -1647 CAAGTTATTCAACTTCATTTCCCCGTCAGTGCTTTAGCT-GGGGACTAAT -1599 opossum -2128 CCTATTATTCAACTACACTTCATTATCTGGGCTTCAACC-GGGGATTAAT -2080 chicken -1775 TGGATTATTCAACCTCATTTCATTGTCATTGCTTCTGCT-AGAGATTAAT -1727 lizard -1811 CCTATTATCCTACCCCA-TTCATTGCCATTGCCTCTGCA-AAGGATTAAT -1764 Xenopus -4058 GGGATTATCAAGCCTCATTTCACT----------CAG--GGCGGATT--- -4024 stickleback -1937 CA--TTATTCTAG-----------------GCCTCTGGC-GGGGATTAAT -1908 medaka -1130 ----------------------------------------GGCGTTTAAT -1121 zebrafish -2227 CATG-AATTATGCAGA-----AGAGATATAGC-----------GATTTGT -2195 human -1720 CAACTATTTATGAGAA-GACA -1700 mouse -1598 AAACTATTTATGGGAA-GACG -1578 opossum -2079 CAACTGTCCCTGGGGA-GACA -2059 chicken -1726 CAGCTGTTTACAACTA-CTT- -1707 lizard -1763 CAACTGCTTGCCCTCA-CATG -1743 Xenopus -4023 CACGGGGTTATCATAAAGATC -4003 stickleback -1907 CATTCAT-------------- -1901 medaka -1120 CATTCAT-------------- -1114 zebrafish -2194 CACTTGT-------------- -2188


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Supplemental Figure 8E. ATOH7 proximal enhancer (proximal CNE) human -751 TTTTTGATACGTAAGCTGCCCACTGAT -725 mouse -692 TTTTTGATATGTAAGCTTCCTGCTGGT -666 opossum -889 TTCTGGTTACAAATGTTGA-------- -869 chicken -884 TTTAAAGTGCAAATGCTACAATGCACA -858 lizard -921 TTTTAGGCAAGCCAACTTCACATCACT -920 Xenopus -515 GTTCTATTAAATATTCTGCAAAGCA-- -489 stickleback -421 TTTTTGTTGTGTGTGCGTGCACTGAGT -393 human -724 ACGGGAAA---------------------AGATAGACCAAGCTCTGCCCC -696 mouse -665 TTGGGAAA---------------------AGATGGACCAGGCTCTCCCTT -637 opossum -868 -----------------------TGGCTGATATGGA--AAATGATTATCC -846 chicken -857 GTGAGAGTTATATATATTTTTTTCTCCTGAGTTGCAACAGATTATCAATT -808 lizard -919 GTGATGTGTCAGGAAAGGTTTC--TGCTGAGCTGCAGAAGGTTATTCACT -872 Xenopus -488 --GGGGGATCTGCCGAGCTTGTTTGGCTGGG-------AGCTGATTAGTT -450 stickleback -392 GTGTG---CATATACAG--------------------------------- -378 human -695 AA---------------CCCCCACCTTGTT-----------ATTCTTTTG -672 mouse -636 CA---------------CCCCCACCTTCTT-----------ATTCTATTG -613 opossum -845 AAGAGGAAAGGTCTTTTTTGTTCCCTTCCCTGACTGTTGTTATTCTTTTG -796 chicken -807 GAAAGGA----------TCTTTTGCTTAAGCGCCAG-----AGTCTAGCG -773 lizard -871 GAAAAGA----------CCTTTTGCTTCGCTGTCGTG----GTTCTTGGG -836 Xenopus -449 AATGGGA----------T-------TTCAGTGGCTG-----GGTT----- -425 stickleback -377 -------------------------------------------------- -377 E4 human -671 GGGACAG-----CC-AGGCCACTC CAGATG GTGTGTTAGTTAGTGTTG -630 mouse -612 GGGACAGTCAGACC-AGGCCACTC CAGATG GTGTGTTAGTTAGCGTTG -566 opossum -795 GGGACAGC----TAG--GCTGCTC CAGATG GTGTGTT-GTTAGGGTTG -755 chicken -772 GGGCCAGC----CAGACACGGCTC CAGATG GCAAGTT-GTTAGGGCTG -730 lizard -835 GGGCCAGCAGG-CAGACACCAGTC CAGATG GCAAATT-GTTAGTGCTG -790 Xenopus -424 ---ACCTC----CAGATATCAGTA CAGATG GCA--------------- -401 stickleback -377 --------------TGTGTGTGTG TGTGTG -TGTGTGTGTGAGTGCTT -345 medaka -235 -----------------------------------------------GTG -235 zebrafish -208 -------------------------------------TGCACTAGTCCT- -199 human -629 GA----CATAGCTTTGGAGAAACT--TCAACAGACTAATGCTGTAATGTC -586 mouse -565 GA----CATAGCTTTGGAGACACT--TCAACAGACGAAGGCAATGATGTC -522 opossum -754 GACAGACATGGCTTTGGAGAGACT--TCAACAGACGAAGGCAGAATTGTC -707 chicken -729 GACAG-AATGGCTTTGGA-AGACTTCTCAACAGATGAAGGCTGTAATGTT -682 lizard -789 GACAGTCAGGGCTATGGG-AGACTTCTCAACAGATGAAGACTGCCATGTC -741 Xenopus -400 GAGCA-CATCCCTCTGCC-AGACT------------GATGCTGCAAACTT -365 stickleback -344 GA------------------------------GAGTGGGTGTGGGTGAGT -306 medaka -234 --------------------------------GCTGTGGAC-GAGATC-- -221 zebrafish -198 --------------------------------GCTGTTG---GAGATG-- -186 human -585 AGGATTTTGCTGTGCGTCGCCC------------CCTCCTC--------- -557 mouse -521 AGGATTTTGCTGTGCGTCGCCC------------CCTCCCC--------- -493 opossum -706 AGGATTTTGCTAGGATTCCC--------------CCCCCCC--------- -680 chicken -681 CGGGTTTTACTGTTGCTTCTTTTTTTTTTTTTTTTTTTGTC--------- -641 lizard -740 AGGATTTTGTTGGAGCAGTTTTTT----------CTTTTTCGGTAGATGT -701 Xenopus -364 AGAGATTTGCTG----------------------CATTCTC--------- -346 stickleback -305 GTGCATT-GGTGTG---------------------TTTCTC--------- -286 medaka -220 TGTGTTTTTCTG-------------------------------------- -209 zebrafish -185 TGCGTTT---------------------------CATT------------ -174 human -556 ---------------CCCGCAGTTCCCAGGGTAGGAAGGTGCTGAGATAG -522 mouse -492 ---------------CCTGCCGTTCCCAGGGTAGGAAGGTGCTGAGATGG -458 opossum -679 -------------CCCCTGCAGTTCCCAGGGTAGGAAGGTGCTGAGACAG -643 chicken -640 -------------CCCCTGTGGTTCCCAGGCTGGGAAGGTACTGGGATGG -604 lizard -700 GTGTGCGTGTGTGCATTTGCCGTTCCCAGGGTGGGATGGTGCTGGGATGG -651 Xenopus -345 -------------ATCCTATATTTCCCAGGGTGGGAATAAGCTGGGGCAG -309 stickleback -285 -------------TACATGCAATATTGAGGATGGGGGG----TGGGGGTG -252 medaka -208 ---------------------ATATTGAGGAAGGGGAGATGATGGAGGTT -180 zebrafish -173 --------------------AATAATAATGGATGTTTGGA--TGGAGTAA -146


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E2 E1 human -521 AAGCCGA-GGGACGCCCGCC CACCTG CCAC CACCTG TTCCCTCCTT -477 mouse -457 GAGCGGA-GGGACGCCCGCC CACCTG CCAC CACCTG TTCCCTCCTT -413 opossum -642 AGGCAGG-GGGACGCCAGCC CACCTG CTAC CACCTG TTCAATCCTC -598 chicken -603 AAGCAGA-GTGCCGCCCGCC CACCTG CTAC CACCTG TTTAGCCTTA -559 lizard -650 AAGCAGAGGGGCCGCCCACC CACCTG CTGC CACCTG TTCTGCTTTG -605 Xenopus -308 AAGCTAA-GGGACGCCTGCC CACCTG CCAC CACCTG CTAAGCCTTT -264 stickleback -251 GGGTCCA-GGGAC-----TC CACCTG CTGC CACCTG TTCA------ -216 medaka -179 ---TGGGGGGGGCGGGCCTC CACCTG CTGC CACCTG TTTG------ -142 zebrafish -145 ---TAAGGGTGGGGC---TC CACCTG CTGC CACCTG TT----AATA -115 human -476 CAGCTCTTT-------GC TATAAA TTCTCCTCCCTCCA -423 mouse -412 GAGCCCTGG-------GC TATAAA TTCTCCTCCCTCAT -369 opossum -597 CAGTCCTCT-------GC TATAAA TTCTCCTCCTCCTC -553 chicken -558 GCAATTT----------C TATAAA GCCTCTTCGAGTCT -522 lizard -604 GCTG-CTGTT------TC TATAAA GGACTTCCTTGGCT -554 Xenopus -263 CCATTCTCTATATATAGA TATAAA TGCAGGGCTTCAGC -243 stickleback -215 ----TCTGCTCCAT--CT TATAAA AGCTGCTCCCTCAC -184 medaka -141 ----TCTGCTGCGT--CT TATAAA AGCTGCTCC-TCGC -112 zebrafish -114 ---CTCT----------A TATAAG CATCGCTCA-TCCT -89


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Supplemental Figure 8F. conserved noncoding element at -38 kb (CNE4) human -38521 CTGTTCAGCATAGAAAAATTAGCTGTGCTCAGGTGGGA----GCTGTT-- -38478 mouse -20778 GGGCCTGGCTTGGAGACATTAGCTGTGCTCAGGTGGGA----GCTGTT-- -20735 opossum -33204 GTGTTCAGCTTAGAAAAATGAGCCGTGCTCAGGTGGGGAG--GCTGTTCC -33157 chicken -14162 CAGTTCTGAATAGAAAAATCACCAGAGCTCAGGTGGGAT---ACTATG-- -14152 lizard -18404 GGGGTGAAGGGAGAAAACAAAGCAGAGCTCAGGTGGGAGTCTGGAGTT-- -18357 Xenopus -12382 ATGTTCACAGCAGACAAATTACAAGTGCTCAGGTGGGATGGTGCATAC-- -12335 human -38477 CAGTAATGTGTTGGTAATAAACAGATTAGTTAATC-AGCCATGATTTATT -38429 mouse -20734 GGGTAAAGTGTTAGCAACAAATAGA-TAGTTAAGC-AGCCATGACTTATT -20686 opossum -33156 CATAACTGTGTTTGCAATCAACGGATTAGTTAATC-AGCCATGATTTATT -33108 chicken -14117 CGGTAATGTGTTTTTAATCAACAGATTAGTTAATC-AGCCATGATTAATT -14167 lizard -18356 C-TAAATGTGTTTGCAATCAACAGATTAGTTAATC-AGCCATGATTTATT -18307 Xenopus -12334 TAGAAATATGCTTCCAAACAACAGCTTAGTTAATTAAGCCATGATTTATT -12285 human -38428 AATGATGAATTAATTATACCTGATTGTGCAGTTCTGCTAGCTAAATGGAG -38379 mouse -20685 CATGATGAGTTCATTATACCTGGCCGTGCACCTCCGCTAGCTAAATGGAG -20636 opossum -33107 AATGATGAACTAATTATACCTGATTGTGCACTTCTGCTGGCTAAATGGAG -33058 chicken -14166 AATGATGAATTAATTATACCTTATTGTGCAGTTCTGCTGCCTAAATTGAG -14069 lizard -18306 AATGATGAATTAATTATCCCTGATTTTGCATTTCTGCTGGCTAAATGGAG -18257 Xenopus -12284 AATGATAAATTAATTATACCTTATTTAGCAGTTCTAATAGCT-------G -12242 human -38378 CTTTAAAGGAGTTGTTTTATTAATCATCAGGGTGCTAATTGCAGAGTCTT -38329 mouse -20635 CTTTAAAGGAGTTACTTTATTAATTGTCATGGTGCTAACTGCAGA----C -20590 opossum -33057 CTTCGAAGGAGTTATTTTATTAATCATCACGGCGCTAATTGCAGACACTT -33008 chicken -14068 CTTTAAAGGAGTTATTTTATTAATCATCATTGTGCTAATTGCAGACACTT -14019 lizard -18256 CCTTAAAGATGTTATTTTATTAATCATTGTGGTGCTAATTGCAGACACTT -18207 Xenopus -12241 C-----ATGAGTAATTTTATTAATCACCTCTGTGCTAATTGCAGATATGT -12197 human -38328 ACAGAAGGATCACTAATTAAACATGC-AACCATATGCAGAACAAG--TTG -38282 mouse -20589 ACAGAAGGATCGCTAATTAAACATGCAAACCGTATGCAGCACGTT--TTG -20542 opossum -33007 ACAGAAGGACCACTAATTAAAGATGC-CGCCGTATGCAAAATAAG--GCA -32960 chicken -14018 ACAGAAGGATCGCTAATTAAACATGC-AGCCATATGCAAAGCAAG--CAA -13972 lizard -18206 ACAGAAGGACTGCTAATTAAACATGC-AACCGTGGGCAGAATGAGGCCCG -18157 Xenopus -12196 ACAGACGGATCCCTAATTATATGCGC-AGCCATATGCAAAAGGGC--TCA -12149 human -38281 GGTTGCTGAGACCTGCAAGAATAAAACATACTCATTTTT--------AAG -38240 mouse -20541 GGTTGCTGAGGCCACGGAAAACCCACCCTCTTCTTTTTTCCCCCAAAGGG -20492 opossum -32959 GGCTGCTGAGACCTGCAAGAACCAGGACTCTTCATTTCC--------CGA -32918 chicken -13971 GGCTGCTGAGCGTGGCAAGAAAAAGAGC-ATTGATTTTC--------CAA -13930 lizard -18156 GGTTGCTGAGAGTGGCAAGAATAGACGC-ACTCATTTTC--------CAA -18114 Xenopus -12148 CGACGCTGAGCTGTGCAGA----------ATGCATGTTC----------- -12098 human -38239 GGAGCCAAAACAAACTTTATTT---TATGCAATATTAGGGAATTGACTTG -38193 mouse -20491 GGAACCAAAACAAACTTTATTT---TATGCAATATTAAGGAGTTCCCTTG -20445 opossum -32917 AGAGCCACCATAAACTTTCCTTCCCAATGACCCGTTCGGCGGCCAGCTCG -30868 chicken -13929 GCAGCAAC---------TCTATCGCAATGAAATATTGCACAGTTGACCTG -13889 lizard -18113 GCACTGA-----AACTTCATTC-CCTATGAAATATTACAGCGTTGACCTG -18070 Xenopus -12097 -----------------------------AAGTCTTAC------------ -12089 human -38192 GCAATGTTAATTAATCACTGCACATTAATATTGTACTTTTTT-CCAGCAGG -38144 mouse -20444 GCAATGCTAATTAATCAC-------------AGTGATGCCCT-CCAGCAGG -20408 opossum -30867 GAGATCCGAATTCCTCCCAGGGAACAGGGCACGGACCCAGCCACCA-CAAG -30819 chicken -13888 GCGATCCTAATTACTCACTA-----------AGACCCACC---CCAGCACA -13852 lizard -18069 AGCATGCTAATTA----TTGC-CAAGAGCC--GCCCAAAC------GCACC -18042 Xenopus -12088 ----TCCCAAACCCCTACTG--------------ACAAC----CCAGAACA -12051


21

Supplementary Figure 9. Concordant spatiotemporal expression of zebrafish 3034-BGnCherry and ath5:GFP transgenes in the developing retina.


22

Supplementary Figure 9. Wholemount and sectioned views of zebrafish larvae showing concordant spatiotemporal expression of the 3034-BGnCherry and ath5:GFP transgenes, driven by 3.8 kb human and 7 kb zebrafish Atoh7 regulatory DNA, respectively. A. Lateral view of double transgenic larva at 74 hpf. RGC projections to the

telencephalon (tel) and tectum (tc) are revealed by the cytoplasmic GFP reporter. B. Dorsal view of 74 hpf larva. C. Lateral view of 53 hpf larval head, showing early stages of transgene expression, with

onset in a ventral patch near the optic fissure (opt fis) and progression around the retina in an apparent nasal to temporal (counterclockwise) direction27-29. Alternatively, the transgenes (and neurogenesis) may be activated with staggered timing from ventral to dorsal, cued by an external signaling gradient30. Expression is stronger (or initiates earlier) on the nasal side of the optic fissure, creating a discontinuity across this cleft.

In addition to the retina, 3034-BGnCherry is expressed weakly in the olfactory (olf)

pits, in a pattern reminiscent of the Xenopus laevis ortholog Xath531. D. Transverse confocal image of 34 hpf head. Expression of nuCherry and GFP overlap

in the retina – in postmitotic cells (closed arrowheads), in G2 cells labeled with EdU during S phase (open arrowheads), and in M phase cells labeled with PH3 (not shown). These results are consistent with the onset of ath5 expression during terminal G2 in zebrafish32,33.

E. Similar transverse confocal image of 48 hpf head, showing overlapping nuCherry and

GFP expression. At the earlier timepoint, ath5:GFP appears to extend further toward the ciliary margin

than 3034-BGnCherry. However, the overall patterns are remarkably similar. The human NCRNA element exhibits highly conserved expression in zebrafish embryos, although the alignment to zebrafish upstream Atoh7 sequences (Suppl. Figure 8a-c) was not statistically significant34-36.


23

Supplementary Figure 10. Model showing potential looping interactions between shadow (remote) and primary enhancers – and the ATOH7 promoter (red lines) – to activate transcription fully. These sequences are bound by regulatory and general transcription factors and the RNA polymerase II holocomplex. The dual enhancers drive very similar patterns of expression in the embryo. Because these appear to be generated using different combinations of transcription factors, the mRNA output should be robust – in aggregate – and is predicted to resist environmental and genetic perturbations, as a canalized trait37. In this model, each enhancer acts with a certain probability in individual retinal cells, creating a dynamic, variegated pattern. The addition of a second, overlying enhancer increases the probability of expression, and this ensures a threshold number of ATOH7 positive cells is achieved for RGC genesis. The combination of two enhancers may also allow refinement of the spatiotemporal expression pattern. The dual enhancers may interact directly and synergistically, to form an active chromatin hub38, or they may function as autonomous genomic modules39 that alternately loop with the promoter40,41. No particular hierarchy or dominance is implied; however, the contribution of each enhancer may be influenced by its distance from the promoter42. In this way, the remote enhancer may be needed to ensure a high-level burst of ATOH7 expression, and initiate retinal neurogenesis. The NCRNA deletion may severely reduce the level43,44 or delay the onset of transcription.


24

Supplementary Note

1. While NCRNA affects the mechanism of neurogenesis, the majority of hereditary human blindness genes are required for retinal cell physiology or homeostasis45-48.

2. The dislocation of Kurdish peoples to northern Iran during the 17-18th century are described in detail by Perry49 and Izady50. Demographic features of modern Kurdish populations in Greater Khorasan are described by Madih51.

3. To better understand the clinical findings in NCRNA, the reader may wish to consult literature regarding the neurological basis of nystagmus52, prevalence of nystagmus in optic nerve hypoplasia53-58 and case reports of sporadic optic nerve aplasia59-65.

The role of melanopsin RGCs in mediating the pupillary light reflex has been investigated in rodents66,67 and primates68,69.

4. NCRNA has not been reported in other large pedigrees70. However, clinically related hereditary disorders may have a similar developmental or genetic basis. These include retinal dysplasia71-73, congenital falciform folds74 and persistent hyperplastic primary vitreous (PHPV)75,76.

5. In NCRNA, the second pair of cranial nerves is missing. Three other Mendelian disorders with reductions in specific cranial nerves have been characterized genetically. These affect oculomotility (congenital dysinnervation syndrome)77, olfaction (Kallman syndrome)78 and auditory perception (cochlear nerve aplasia)79.

6. The NCRNA deletion represents one of few human mutations in the proneural class of bHLH genes80. There are three other examples, affecting development or function of pancreatic islets (NEUROD1)81, enteroendocrine cells (NEUROG3)82 and brainstem respiratory centers (ASCL1)83.

7. To better understand the tractional basis of NCRNA pathology, the reader may wish to consult literature regarding retinovascular development in primates84 and mice85, and the elevated risk of juvenile detachments in other human congenital disorders of the posterior retinal vasculature, including ROP, PHPV and FEVR86-89. Partial congenital retinal detachment has also been reported in patients with less severe optic disc anomalies90.

8. To better understand the connection between RGC, retinal astrocyte and vascular development, the reader may wish to consult literature regarding retinal angiogen-esis85,91 the origin of retinal astrocytes92,93 and specific RGC signaling factors that mediate retinal vascularization94-98.

9. To better understand the secondary keratopathology in NCRNA disease, the reader may wish to consult the literature on chronic corneal blood staining99-101.

10. For more background information on optic nerve hypoplasia, the reader may wish to consult literature on the clinical features, syndromic associations and epidemiology of ONH53-55,102-105.


25

11. Mutations in Pax6, Pou4f2, Rx, Hesx1, Otx2, Six6, Chx10, Sox2, Dcc, Ntn1, Cyp26a1-Cyp26c1 and Rpl24 have been associated with optic nerve hypoplasia or aplasia in humans and/or mice106-117. These genes encode transcription factors, signaling molecules important for axon pathfinding, retinoic acid catabolic enzymes, and a 60S subunit ribosomal protein.

12. Extrinsic factors Fgf (fibroblast growth factor), Shh (sonic hedgehog) and Gdf11 (BMP growth-differentiation factor) have been shown to regulate RGC genesis or Atoh7 (ath5) expression22,118-122.

The apparent wave of neurogenesis123 and Atoh7 expression28 in zebrafish and chick retinas has been attributed to propagation of a Hedgehog signal27,124 from nascent RGCs, but may have a cell-intrinsic basis30,119.

13. For a more complete understanding of Atoh7 transcriptional regulation, the reader is directed to functional studies of orthologous upstream sequences in mouse, Xenopus, chicken and zebrafish7-11,13,32,33,125-128.

14. Although Atoh7 is necessary for ganglion cell development, its enhancers cannot be used to label RGCs directly, since Atoh7 is transiently expressed in a subset of multipotent precursors, only some of which develop into RGCs129,130.

15. References for specific Materials and Methods are:

Multiple displacement amplification (MDA) of genomic DNA131

pBGnCherry plasmid vector132

Rat polyclonal β-galactosidase antibody133

Tol2 transposase-mediated transgenesis in zebrafish134,135

PTU (1-phenyl-2-thiouracil) inhibition of zebrafish pigmentation136

ath5-GFP zebrafish transgene12

Labeling proliferating cell nuclei with EdU (5-ethynyl-2’-deoxyuridine)137

Multispecies DNA sequence alignments138-143


26

Supplementary References

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2. Zietkiewicz, E., Richer, C., Makalowski, W., Jurka, J. & Labuda, D. A young Alu subfamily amplified independently in human and African great apes lineages. Nucleic Acids Res 22, 5608-5612 (1994).

3. Prasov, L., Brown, N.L. & Glaser, T. A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina. PLoS ONE 5, e12315 (2010).

4. Ghiasvand, N.M., Fleming, T.P., Helms, C., Avisa, A. & Donis-Keller, H. Genetic fine mapping of the gene for nonsyndromic congenital retinal nonattachment. Am J Med Genet 92, 220-223 (2000).

5. Cheng, H.L., et al. Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc Natl Acad Sci U S A 100, 10794-10799 (2003).

6. Duboscq-Bidot, L., et al. Mutations in the Z-band protein myopalladin gene and idiopathic dilated cardiomyopathy. Cardiovasc Res 77, 118-125 (2008).

7. Hutcheson, D.A., et al. bHLH-dependent and -independent modes of Ath5 gene regulation during retinal development. Development 132, 829-839 (2005).

8. Riesenberg, A.N., et al. Pax6 regulation of Math5 during mouse retinal neurogenesis. Genesis 47, 175-187 (2009).

9. Willardsen, M.I., et al. Temporal regulation of Ath5 gene expression during eye development. Dev Biol 326, 471-481 (2009).

10. Hernandez, J., et al. Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating retinogenesis. J Biol Chem 282, 37894-37905 (2007).

11. Skowronska-Krawczyk, D., et al. Conserved regulatory sequences in Atoh7 mediate non-conserved regulatory responses in retina ontogenesis. Development 136, 3767-3777 (2009).

12. Masai, I., et al. N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites. Development 130, 2479-2494 (2003).

13. Del Bene, F., et al. In vivo validation of a computationally predicted conserved Ath5 target gene set. PLoS genetics 3, 1661-1671 (2007).

14. Nelson, C.E., Hersh, B.M. & Carroll, S.B. The regulatory content of intergenic DNA shapes genome architecture. Genome biology 5, R25 (2004).

15. Driever, W. & Nusslein-Volhard, C. The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo. Nature 337, 138-143 (1989).

16. Hanes, S.D. & Brent, R. DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell 57, 1275-1283 (1989).

17. Treisman, J., Gonczy, P., Vashishtha, M., Harris, E. & Desplan, C. A single amino acid can determine the DNA binding specificity of homeodomain proteins. Cell 59, 553-562 (1989).

18. Dorval, K.M., et al. CHX10 targets a subset of photoreceptor genes. J Biol Chem 281, 744-751 (2006).

19. Vinothkumar, S., Rastegar, S., Takamiya, M., Ertzer, R. & Strahle, U. Sequential and cooperative action of Fgfs and Shh in the zebrafish retina. Dev Biol 314, 200-214 (2008).

20. Raible, F. & Brand, M. Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development. Mech Dev 107, 105-117 (2001).


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21. Roehl, H. & Nusslein-Volhard, C. Zebrafish pea3 and erm are general targets of FGF8 signaling. Curr Biol 11, 503-507 (2001).

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SUPPLEMENTARY TABLE 1. PCR PRIMERS FOR ATOH7 MUTATION SCREENING size anneal REGION amplicon [bp] forward primer [5’-] reverse primer [5’-] temp MA 5’ flank L 820 CCTGTCCCTGTTCACCCTAAGG TCACCAAAGGACCCCAGGAGG 52 J 684 TCCAGCAGTGTGCCAAGTGAC GGAACAATGAAGCTGAGGTCCAC 52 KJ 530 CCAGAAGACAGAGTCAGATTCCGC GATGGAGATAAGGAGATGGCAGG 52 K 757 CCAGAGTCTCCTGACTGCTACACTC GGCTCACACCATCATTTCAGCAC 52 M1-4 897 GACAGGGTCTTACTATGCTGC CTAACCTGCTCTAGTACATTTC 55 M3-2 1127 GGGCAAAAAGAGTGAAACTTTGTC TGCATGGAGCCACTCAGCTCTG 55 RE 426 GAGAAACATTCCACAGAGCAGCAC CCGTGACTTGCTTTCCCCAC 55 D 1163 CCCAAAGTAACAGAGCTGAGTGGC GCTTGGTCTATCTTTTCCCGTATCAG 55 1X 5’ UTR F 563 GTTTTTGATACGTAAGCTGCCC GACCAGGCTGATATCTCTTCACTTGCC 55 tata 514 ACCACCTGTTCCCTCCTTCA CTGAGCAAATAAGTCATAAACAAAG 60 4X CDS A 269 ACGCGCAGGTCCTGAGCGGG CCACTGGGGAACCACCCTGCGTAA 60 4X B 239 GAGCGCCGCCGCATGCAGGG CTTCGCGCCCGGGAACGGGA 60 2X C 242 GGTCTCCACTGTGAGCACTTC TCGAGGATTGCATCCTTAGAATCC 60 3’ UTR G 589 AAGTAGCCCAGAAGCCTCCG GCCATCAGGAAACTGGCAAGG 55 3’ flank H 487 ACACATATACAGGAATACACTTC ATTACACACCAAACGGATTTTG 55 PCRs were performed using Platinum Taq [Invitrogen] or EXPAND high fidelity [Roche] reagents, with 200 nM each primer, 1.5 mM MgCl2 and 200 µM dNTPs. MA, Masteramp concentration [Epicentre]. Cycling conditions were 95°C x 5 min, followed by 40 cycles of [95°C x 45sec denaturation, 52-60°C x 45-60sec annealing, 72°C x 45-90sec extension], followed by 72°C x 7 min. The amplicons extend from -5.53kb to +2.37 kb relative to the ATOH7 promoter, and are shown in Figure S1.

Nature Neuroscience: doi:10.1038/nn.2798

1

SUPPLEMENETARY TABLE 2. PCR PRIMERS FOR NCRNA CANDIDATE GENE SCREENING

size anneal

GENE exon [bp] forward primer [5’-] reverse primer [5’-] temp

LRRTM3 1 733 ATGCCCATAATGTTCTTCACC GTCTTAGAGCAGAGCAAGGTAATC 52

2a 1069 GTGGCATGTTTCCTTGTTTAAC TGTGAGCTTGTTGGAATCCAG 52 2b 936 CAGCCTTCAGAACCTTTACTTG GCGTTCCCTTGATAGTCACC 52

3 372 GGTCAGCACTTCAGTCTCTAAATC CCACAATGAACACATCTTCCTC 52 4 3110

CTNNA3 1 318 CATGTTGCTTTAAGAGCACCCATC TCCCCCAAAGTTATCTAACCGAG 52 2 273 TCACCACAGGAATAGGCTATCTGTC CTTGTTTCTCTCTCCTTTGCGTTG 52

3 473 GATTTTGTAGCCTGGTCCTCTGAG GCTAAAAGAGAATGTGGGGGAAAC 52 4 397 TCTGGTCTGTAATATCCCAGGACTG GCTCCTCACAGCTCTGCAAAAGAC 52

5 477 GTTGAGCACAACTTTCTTGCAGG TCGACGCCAGGTTCAGAGAATG 52

6 334 TCGACTCTTCGTGTTAGCAAGTTG GCACAGCACAACCTGTATCTGCTC 52 8 730 GGATCTTTACACATCTGCTCTACGG TTCATTGGATTCCTTGGCAGAG 52

9 527 CCGAGGCAGTAAAACACAGTTCTC TCTCATCCCTAGAAATTCCCAGG 52 8 3529

DNAJC12 1 435 GGCAGCACTGCAATAATAGACAC GTTAGGTTTGTAGGCAATGTGAC 52 2 264 GTCACTGCGCCTGGCATGAC CCAATAGTGCAAAGAAAGAATACGG 52

3 444 GTAAGAGCAGTTGGATAGTTTTGG GATTGAAAGAAATGTAGTCACAGGC 52 4 397 GGTCAGGTGTTACCACATATCAAAC TGGCACTGCTGTGCAGATTG 52

5 323 CCCCAAGCTGTTTTACTATAACAAG TGAGAACTGATTCAAGGATGGAG 52

5 1863

SIRT1 1a 499 GTGACCCGTAGTGTTGTGGTCTG GCCCATTGTCTCCTTCCCCAGC **57 1b 361 GAGCAAGAGGCCCAGGCGACTG AGAGCGAATCCACTGGCAGCTC **57

2 366 CCAATGAACAGTGCAAGCTGACC CCAGCCTCCGCAACAAGAG 55

3 466 GCAAAAAACCCTCACAGAATGC GGCAGTTCCCAAACATCCATTATC 55 4 547 AATGCTTAAAAGGCTTAAAATTGGC GATCTTACTCCGCCACAGTAGCAG 55

5 472 CGACTTTGTCTTTTTCTTCAAGGG TCTCCACTTCTCGATGGCAGTC 55 6 360 ATTCTTCATCCCCTAGATTCTCCTG GGGGTAAAAAAGGATTTTGAGGC 54

7 413 CATTGACAGTTGCTGTTTATCC AGCGTCTATTATACAGACCCAC 52

8a 559 AGTGCATGGGTCTTTTTTGGG CCATCTGTTCAGCAATACTTTCAAC 54 8b 283 GGTTGTATGGAAGAAAAACCACAG CGATTAACCTGCCGAAATAGC 54

9 439 CCACACTTCATTCCAGACTGCTC TGGAACAATTCCTGTACCTGCAC 54 11 4771

HERC4 1 403 CGTGCTCTGCGGTTCACTTC TAGGGGAAGCCCAAGGTGAC *60 2 217 GGCTAAAGAGTAATTGGGAGAGTCG GACATCTTTCCTCTGCCCAAAAAG 52

3 470 AATTATTGTAGCCTAGCACTTG CAACTAGAGAAATTATACGATGATTC 52 5 493 CCACAATGAGAATTACGTTTGGAAC AAGGACTCAAAGGGGAGAAATAATG 50

7 517 CCAAGATGTGTTCTTTTCACCCC CAAAATCCCAAATTCCTAGCAGTG 50

8 478 CTTTTGATGATAGGCAGAATGTC AATAACATCTCATCCCCACC 55 9 428 AAGCTGTAGAGGAAACACATC TTCATATAAAGCTGAGAAATTCAGC 52

10 452 AACGGTGAAAACATCATTACCTGG GGTATTTGGGGGCAGAATGTG 50 11 481 CAGCCTGTCTATGTGATGCAG TGCTGTTTTCACTTAACAATGTATC 50

13+14 607 ATACCATGAAGTCCATGAATGG CTATCTCCACAGGAAACCTTC 53

15+16 763 AGGCATCCCAGATTAGGCTC CGTAAGAGTCTGTGAAACTGCAAAG 55 17 602 CACCATTTCATATTTGCTGTAGTGC CGATGAGGGAAAGGAATACG 53

18 489 TTTCTGGGGTTTTCTCAGG CAAAGTCAGGTAAGACCAGAGC 53 19 420 GTTGACAGAGTGAGACTCCGTC GGTTTGGCTTTGGTAAAAGAGTAG 50

20 256 GGGGGTGTATTTGATTGTAGATG TTTGGCTCTTCTATTACTCAGGAG 50

21 321 CCAGAACCACATGAGTGAATAAAC CTTTGTGTACCTTCATTACTGATCC 50 22+23 756 CAGTAGCTTTATTGTAGCATTGCTG TTTGACAACTCCAAGTCCAAAG 50

24 593 GGGAGACAGTAATGAGGATGAAC GCTGGTCTCAAACTCCTGAC 55 25 1595 GGAGGTAAAAAGTGGGGAAG TTTAAGTCACAGAGGCTGGTATG 55

26 362 GTAGGATTTCAAACTAGAGAGTACC GTTGTGACACGTTTTCTACG 50

27 422 GCAGAAATTGTAGGAGCACTTG TGTGGAGTTTGAAGGGTAGAGTG 50 28 263 CTGGACCATGTTAGCTTTATGTG GAAGATTAAAACAGGGCTTTGG 50

29 915 TGAAGCTCTGCTGTAGCGAC GTGATTTAGGTTTGCACAAAGACAC 55 23 12303

2

SUPPLEMENTARY TABLE 2 (continued). PCR PRIMERS FOR NCRNA CANDIDATE GENE SCREENING

size anneal


MYPN 2 1000 TTCCCTAGAAATGATCCAAA TGGTAGATGGAAAGCAACAT 60

3 542 GCTGTTCCTGCTTTCTCACC CCTCATTGCCTAGAAGAGCG 52 4 319 CGGCGTTACTGAGTTCATTTC TTTTTGTGGTCAAACCCTGG 60

5 285 CCTGTAAGCAGTGATGCCATT CATGCAAGATGGTCATGGTC ‡60 6 346 TTTTGGTTTGGGATGCATTT AAATCTGGGACTTGAAGCGA 52

7 372 TGGAGACGGCATCTTTTTCT ACAGGAGTATGATTACAGGTG 52

8 408 TGCTGCCTTTCTGTAATATCC TGATCTCAAGTAATCCGCCC 52 9 300 TAGCCAGCTTTTTATATTGACT AATGGAAACACAAAATCTGC ‡60

10a 376 CTAAAGCAATCCTCCCACCA TTGGTGTTGTTCCAGTAAGAC 52 10b 939 TTACAACAGCTTCATAACCAAG ATTACAGGCATGCACCAACA 52

11 850 ACATTGTTTGAAAGGTGAGG CCCTGTTTCAGATCAAGCTA ‡60

12 297 TGTCATTTCAACCACTCTGA GAGGACTGAATCAAGCAAAA 52 13 368 TGGCTTCCTCAATTGTACTGA AGACCTTCTTGAAGGCACTG ‡60

14 334 CCTTTCCTCACCCCAGACCC CTCAGCAATCCTTAGTAACTCGCAC 52 15 239 GCCAAAATTTCACGGTGTTC CGATAGTGGGCAGGTGCT ‡60

16 271 CCATGCCTAAATGCCTATT AAAATGAATGCAAACAAATACA 52

17 379 GGTGCCAAGCAAGGATAAAG TGAAGGAATTCTGGCAGAGG 60 18 463 AGATCTTGCCACCACACTCC GCCAGACTCAAATAGCAGCAG 55

19 292 CCTGGAACCCTAAATTTGACA CCTTGCCTGACCCATTTATC 52 20 924 GGTGAGGACAGAATGCACCT TGCTTTGGCCACAAAATAAG 52

20 9304

PBLD 1 386 AGACCAGAAAGCTAGGGAGAGTACG AAGGGATGAGGGTGGGTAGTGAAG 52

2 428 GGCAACAGAGCAAGACTCCATC GGCAAAGAACACAAACCAAAGAGG 52 3 392 GCATGGGTTTCTGAGAACCAATC GCACAATAATTGCTTGAACGCAGG 52

4+5 530 CCCAAACCTCGCTCCATCATAG GGAAAACTGGTCAACCTTCTCACC 52

6 349 GGCTCACAGTGGATGCTCAATG GGAAGGAAGAACAAGTGGGAATG 52 7 461 CCAGAGTGTTTCTTTCCCTGATAGG AGGAACAAGGCAGCTCAAAACTC 52

8 403 CATAATTCTACACCCTGCTGTGGC AGGCAGAGGTGAAGGCTGTTTG 52 9 317 TTCTTGAAACTGATCTGGGAGGAC CTACGCACATTTGCTAAAGGCAG 52

8 3266

HNRPH3 1 380 TTGCTCGTTCCCTCCCATCC CAAGTCCTCCCATCGGCAAG 52

2 564 GGGTCAGGTCTCATTGCCTTTTC CACGGTTCATTTCATCACAATTCTC 52 3 347 TGGGGTGATGGGAAACTAAGC CCCATTTAAGCAATAGTGACCCAC 52

4 436 GAAAAACTTACTGCATTTTGCCCAG TGTGCATCAGTCAAGTCCCACAG 52

5 476 CCATCACGTTGACTGCTTTTTTCAT AGCCATGTCCTCCCATACCTGG 52 6 432 TGATGGCTTTGATGACAGAATGAG AAGAATGCTTAAAGATTTAGACACC 52

7 389 TTAGTCTGACAGGGCAGTGGAAG CCCACAGAAAGCCATTATAGAGGTC 52 8 382 TGACCTCTATAATGGCTTTCTGTGG CCCATTCCCATTCTTCCAACTG 52

9 448 ATTCTACTCCTGGAGGCGGCTC TGCTTTCAGTACATCCCACGC 52

10 266 GATGGTTTGGGTGGTTATGGTAAG GACCAGATGCTGTTGTCAAGACTTG 52 10 4120


3


size anneal


RUFY2 1 513 CCTTCACGTTCCTTTCCTTCTCC GCCACTATGCCTCAGAACCCG **60

2 354 CCGGGTTCTGAGGCATAGTGG CAAGGAGTACCAGGGCCAGTG **60 3 355 ACCAAGGAACAGCAAACTACTG TAACCCATTCTCAAGGGCAC 52

4 515 CTCCAGATCCACTGGGATATG CAATTTCCTACAATACAAGGCAG 52 5 409 AGCTCTCAGGGATTGGTTTG TCTGTCTCATTTTGCTCCTCC 52

6 322 TGGCTTCCATTTCTGTAAGGTAG CCTGTTTACAACTTCAAATGACACC 52

7 443 AGTTTGCACAGTTCCTCCTTG CAGGTATAACCAAAGGTCCCAC 52 8 337 GCAAGTAATTTATTCAACAAAGG GAAAGGAATACGGTAAGCTG 48

9 291 GGTGCCTGTCAGTAAACATAG TTGCATCACAATGATGAGTG 52 10 478 ACAGACCTTTTGCTAGTTGTG CTACCTGTCGTAACTGAGATTC 52

11 463 TTCAAGCAGACAATCTCGTG ATCACACCACTGCACTCCAG 52

12 449 GCTGGGATTACAGGTATGAG GGGCTCATCTCTTAAAAACC 52 13 397 ACAGTCCCATAGCAAAATGG TGTGGCAGGTGTAGGAACTG 52

14 503 CCATTGCCCTGTCATGTTGG TGCACCCACTCTGCTTGATG 52 15 517 GTGGCTCTTTCACGTTTGAG TCTCCTAGAGCAAGAAGACAG 52

16 481 AAAACCCTGTGCTGCTGAAC CCAAAGCTCTTTCCTAGCTG 52

17 312 GCTAGGAAAGAGCTTTGGAC GCCTAATAGATTTGGCACAC 52 18 264 TTCCACTAGCCAGAGAAGGC GATTCACACAGGGCATTACC 52

19 431 TACTTAATCCACATCCCACC GTTGTCAGAGCAGGCATTAC 52 20 323 AGGGAGATAAGAAGTGAGTCC TAAGGAGAGCTGTCTGGTTAC 52

20 8157

DNA2L 1 566 AGAGAGAAGGCAGGAGCGTTTAG AGGCTCTGGCTGAATGTGGC *52

2 528 GTTTGAAATGGCATTACTTCTGGG TTAGGCTAAGATCAAACCTACTCC 52 3 411 TTTTGAAGCCTCCCTAATACG CTGAGCCCATTTTCCTCATC 52

4 424 GTGCATTTTGGCTATGGTGG AGTTCCTGTTGAGCAATTTCAG 52

5 339 CTGACGAACAGTTGGGTTGC CAACACACAGAATGGCATCC 52 6 411 CAGTCAGAGCCAGCAGATGTG GCTTTCAGTGAGCCAAGACC 52

7 360 CTTTACCAATTTGGCTCATGTTAC AGTGCTGTGAGTAGATTGTCAGG 52 8 446 GCTAGTCTTGAACCCCTGACC TGATGTTATCAATGCCCGTC 52

9 616 AGTTGTTTGGGGGATAGTCG CATAGGATACACAGGAATGGTGC 52

10 342 CAGATGAACAAGTAAGTTGTAG AAAAAGTAACATTTCATTACC 48 11 400 TGTGACTGAGGGCTGGTAGC GTGGAAGAACAGAACTAAGGTAGG 52

12 322 CGTTTGTCAGGTTTGTAACAATGAG CCATTTCCAAAGAGTCTTGGTC 52 13 410 ACCAAGACTCTTTGGAAATGG CAACTGTTTGTGCTTCAGCTTTAC 52

14a 241 TTACTTGGCTCAGCTCTTAGATATG CCTTCTGAATCTGACCCAAAC 52

14b 273 TTTGTTGACCAGCTATACACACTC ATTTCCCAGAGTACAGTTTCTCC 52 15 424 GGATTCCTGATGCCATAGAAC AGCTTTCACTCATGCCAAGAG 52

16 338 CCTGGTGCTAAACCGTGAAG CCAGCTTGCCCTCATAGGTC 52 17 425 GCTGGAGCAGAATAAGAGTGC GGCATGTGCTAATGAAAACTGAG 52

18 323 TTTTTCATCTCCAAGTCGGTAG AAACAATGGGGCTTCAATCC 52

19 317 AACATCAGTTGGGCACATAATG CCCAAATGAATCAGTAGCAAACAC 52 20 273 GGTGTCCATTGTGTAGGGATG AACAGTGCAATCAAATGTGACC 52

21 344 ATTCATTGAATGCCATAAAGATCC TCAGACTTTTCACAGTTTTCGG 52 22 8533

SLC25A16 1 567 CCATCTCACTGCCTACCGTTTC CCAGAGTCCAGGAATGTCATAGAGG *55 2 252 GGCTGAACAACTAATGATTTCCATC TACACCCCCAAACTTAAATTCCC 52

3 310 ATTCATGTAATCCCTCAAGC TGCATGATATTCAAGAACGC 52 4 326 GCCAAGCTGTATTTAATTGTGCTC CTGCAACCTGTGCTTTCCCC 52

5 450 TGACCCTTCACTTACTAGGTTG CCCTGAAACACAAAGATGTG 52

6 412 CATCTTTGTGTTTCAGGGTG ACAGCTGGCTCAGATATTAAG 52 7 434 GCAGTAAACTTCAGTGGTGC GAAAATCAGGACTGGGTAAC 52

8 298 GACAGAACAATAAGAGCAGTAACAC GGAGATAAAACGGAAGATACAGAC 52 9 312 GGGCATAGAGGTCATCATTTAATC CCAGTGGCTCTTGTGACAGG 52

9 3361

TMEM14D 1 483 GGTAGTCTCCTTTCTGGACTG ATATCAGATGCAAAAATGCTGCAC 52

1 483

4


size anneal


CXXC6 1 685 GCGTTTTGTCTCTCGCTCAACTG CGGGGGCATTGTTTATCTCC 58

[TET1] 2a 1154 GTGAATATTCTATTTCAAATAGGGG AGACCCACCAAGAGCCAAGC 52 2b 1234 ACCCCACCTCCAGTCTTAATAAGG GCTAAGGCATCACTGCACAAGAC 52

3 389 GCTCTTTAGGTTCTGCCTAGCAAAC TGCTAATATGCACATTATGCAGCAC 52 5a 1457 AGGTGTGAGTCACTATGCCTAGCC TGGGTTGCATCCTGACATGATAG 52

5b 1421 TGATTTGCATCAGTTGCCACC TTCCACTTGAGCGGGTTTACC 52

6 473 TCGTTAGGGGCAGGAAGAAAG TGTATGATGGGTGGCTCAGTAGG 52 7 218 TGTTCAAAGCCAAAGCCTTCAAG CAGCTCTGGAATAACAAGCTTC 52

8 425 AATCATGGAGAACATCAAAAGG TTTTAATTCCACAATACTCAAATCC 52 9 498 TTTAGGCATCCATCCTTCTAAAGC GGGTAGTAGAGCAGGAGGAGGAAG 58

11 248 GTGCATTAACCACCATCAGC ACAAAGCAAGACCCCACCTC 52

12 485 CCTGAATGTTGCTAAGCTACACC GCATTTAATGAAGACCTGCACTAC 52 13 513 GCAACAACACGAAGGTAGGAATAGC ACAAATAAGGCAAGGCAAAGTCC 52

14 1305 GAACAAAACCAACCAACACAACATC CACAGTTAAGAATACCCACCCCG 52 14 10505

CCAR1 1 220 CAGAACGTCAGCCAGTAAGCG GCCCTTCCACAGATTCGACAAG 49 2 343 CTGCTGAAATCTGCGAAGACG AAGTGCTAGGATAACAGGCGTGAG 49

3 512 TCATACGTGGTGCAAATTACCG TTCTTCACTACCTGTAAAATGAAGG 49 4 356 TTTCTTTGAATCAATGCCACACC TCCCTCACCCTTGGAGACGTTC 53

5 372 TGGGCAACATAGTGAGACCTCG GCTGAACCTACAAGGAGAAACCATC 55

6 603 GTGGTCAAAGTTTGAGGAATGCTG CGACTTGAGATTGCACAACTGTGTC 55 7+8 662 AAAGAGCTTTCCAGGCTTCC GTTTTCTTGAAATCCCTGCC 49

9+10 841 ATTTTATCAGGTAATGCCTTCG GAGGGGCTGCCTTTTTCAATAC 50 11 729 AGTTCCAACTGAGGAAAACCTGC CTCAAGAGATGAGGCAAAGAGAGC 50

12 233 TTAGACTTTTGACAGGATTGAA GGGCTTTAGATTTTATGTGTA 49

13 405 ACTCTTCCTTGACTGACAGATACC CCAGGGTTGCTAAAATAAGCCG 49 14 563 AATGCCTTGGGGGAACATATTC GCTGAAAGCTGGATTACCAGTCG 50

15 319 TGGCTTTGATTGGCTGATGAG AATAAATAAAATCCATCCTTATC 48 16 337 GTTTGTTTCCTATTTCAATATCC CGTATTCCAAGAACACCCTA 48

17 556 GAGCTTTCCTCCCTTCTCAACG AGCCACCATCATGCCAATGC 55

18 294 ATTTGGCTAGGTTCATGTTG AGCAGGTTGTTATTACAGACC 53 19 502 TGGCTCACGCCTATAATCCTAGC CGGGAAATTTGTTCCGCCAAATTG 53

20 204 TAATGACTTTTTTGCCACTG GGACTAATAAACTTCACAACTGTG 49 21 309 TTGCTATCTTATGCACAGTCTC ATTAACTTTTCACTGGAAAGG 49

22 286 CAAGAAATGTATTTTCATCTCTG TGGAAGTAATAATCTGTTTCCTA 45

23 348 AACCATGAAGAGTCTGAGTCAT ATTAACATATTCAAAGCTGACC 45 24 474 ACAGACATTGCCATTTTATG TTTACTAATCTCAGGAAGGTCAC 49

25 175 TTTCCTTCTCTATTGTCTTATAGG AAATCAAACATGCATCACCTTC 42 23 9643

TOTAL 178 82948

PCRs were performed using Platinum Taq [Invitrogen] reagents, with 200nM of each primer,

200 µM dNTPs, and 1.5mM [or 2mM]‡ MgCl2, and were supplemented with 2.5 U EXPAND high

fidelity polymerase [Roche]. Reactions included *1X or **2X Masteramp [Epicentre].

Cycling conditions were 95°C x 5 min, followed by 40 cycles of [95°C x 45sec denatura-

tion, 42-60°C x 45 sec annealing, 72°C x 60sec extension], followed by 72°C x 7min.

Only the proximal nine exons of CTTNA3 (!T-catenin) are located in the nonrecombinant

[NR] interval for NCRNA. The LRRTM3 gene (leucine-rich repeat transmembrane protein) is

inside CTNNA3 intron 11, just outside the NR interval, and is transcribed from the opposite strand.


SUPPLEMENTARY TABLE 3. PCR AMPLICONS FROM THE 74kb PBLD-ATOH7-MYPN INTERGENIC REGION ATOH7* Fig 3 size AMPLICON [kb] label [bp] forward primer [5’-] reverse primer [5’-] NCRNA PBLD [>] -52 14 -37 818 CATCAGGGTGCTAATTGCAGAGTC TCTAACTTGGTTGAGCGTGAGTAGC + 23 -29 a 886 CAGGAAAAGAGCCAGGGGTAAG GGGACCTATGGGACAAGATTCTCAC + 27 -25 b 379 CCCATACTCTGCAAGAGAGCC GAGAAGAGAATGGAAGAATGTACC + 28 -24 c 162 TTCCTCAGAGGCAACCACTC GATGCAAAACAGTGTGATTAGTAGG + 30ref -22 468 TGCAAGGACTTTGTGGCTCTTG TTGATGAAGGGCTGCTAAGGTC - 30 -21 d 557 CTTTTCCACAGTCCTGCATCATTC CCCAGTGTTTCCTAGCTTTGTTAGG - 31 -20 e 199 TCTTACTCATTGCTGTCCCG CCAGGTAATCCAGCCAGAGG - 33 -19 f 138 GCTTCCATGATGAATGTTTTGGAC AGACCCAGAAATCACCGATG - 36ref -16 502 CAAAATACCCAAGTGGCTTCCTC GCTTCAGCTCAGCTACAAAGTTACC - 36 -16 g 514 TCTTCAAATGCCAAAGGTCCAC TGGGGGATGATTTCCATGC - 37 -15 h 215 TGGTAGGTAACATCAGGACTGG TTCAGAACAGGAGCAGCGTG + 39 -13 i 228 CAGAGGCAGGTTATTCACAGAC TGACCTTCCTGCTTCTGCTG + 40 -12 275 CCAACGGAACTATGAGTGTTTG AGTCTGTCTTAAAACATTTCAGTGG + 44 -8 j 744 TGGCTTCCAGTTTGGGGTTC AACCTACCGTGAGCAGTAAACCAG + ATOH7 [>] +1 k 242 GGTCTCCACTGTGAGCACTTC TCGAGGATTGCATCCTTAGAATCC + 61 +9 1162 GTCCATCACATCCTCTAAATTGCC TGCCCACTACTTTGTCCTTCCTTAG + 68 +16 591 CTGGGAATGATTCTTCCTTGTACG AAAGGTCTGAAGGGATGCAGATG + MYPN [<] +22 PCRs were performed using Platinum Taq [Invitrogen] reagents, with 200nM each primer, 1.5 mM MgCl2 and 200 µM dNTPs, supplemented with 2.5U EXPAND high fidelity polymerase. Cycling conditions were 95°C x 5 min, followed by 40 cycles of [95°C x 45sec denaturation, 52-55°C x 45 sec annealing, 72°C x 60 sec extension], followed by 72°C x 7 min. The PCRs and 74kb intergenic map are shown in Figure 3b-c. The amplicons are oriented in the same direction as ATOH7 transcription [10q telomere to centromere] and listed in order from the 3’ end of PBLD [chr10:69,714,052 in NCBI36/hg18] to the 3’end of MYPN [chr10:69,640,048]. Both flanking genes are transcribed toward ATOH7. The PCR products shown in Figure 3c are indicated with labels. The NCRNA deletion (blue box) was revealed by absence of specific PCR products. Underlined primers were used in the diagnostic triplex PCR. *Approximate distance from the ATOH7 promoter.


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Supplementary Material for - Nature Research€¦ · Supplementary Material for Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease. Noor