Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia

Abstract

The strictly regulated expression of most pleiotropic developmental control genes is critically dependent on the activity of long-range cis-regulatory elements. This was revealed by the identification of individuals with a genetic condition lacking coding-region mutations in the gene commonly associated with the disease but having a variety of nearby chromosomal abnormalities, collectively described as cis-ruption disease cases. The congenital eye malformation aniridia is caused by haploinsufficiency of the developmental regulator PAX6. We discovered a de novo point mutation in an ultraconserved cis-element located 150 kb downstream from PAX6 in an affected individual with intact coding region and chromosomal locus. The element SIMO acts as a strong enhancer in developing ocular structures. The mutation disrupts an autoregulatory PAX6 binding site, causing loss of enhancer activity, resulting in defective maintenance of PAX6 expression. These findings reveal a distinct regulatory mechanism for genetic disease by disruption of an autoregulatory feedback loop critical for maintenance of gene expression through development.

Figure 1

Aniridia Subject ID 1230P Carries a Mutation in a Remote, Ultraconserved PAX6 Regulatory Element (A) Map of the PAX6 locus on human chromosome 11, displaying the exons of PAX6 (black rectangles, top strand) and adjacent ELP4 gene (black rectangles, bottom strand), whose introns contain long-range cis-elements (blue ellipses) for PAX6 (including the distal regulatory region [DRR]). The SIMO element (orange ellipse), located 150 kb downstream, is deeply conserved with strong sequence similarity across vertebrates. The alignment shows the centromeric part of the element. The mutation changes a 100% conserved residue in a highly conserved sequence block with strong similarity to a PAX6 binding site. (B) Visualization of evolutionary sequence conservation by percentage identity plot (PIP). Green boxes highlight presence of the element and purple indicates its absence in the zebrafish pax6.1b locus. Fragments characterized in the EI-Z, SIMO-LacZ (SIMO-Z), and SIMO-GFP transgenic reporters are indicated. (C) Eye phenotype of the affected individual and his unaffected parents. Slit lamp and close-up photographs of the eyes of subject ID 1230P show complete absence of an iris (arrowheads) and presence of lens cataracts (arrows) in both eyes. Ocular fundus photography and horizontal optical coherence tomography (OCT) in comparison with a normal eye reveal absence of a foveal depression in the subject. (D) Sequence traces from affected individual and parents show the de novo heterozygous G/T transition in the element. A consensus PAX6 paired domain binding sequence is shown in alignment above the sequence traces.

Figure 2

Characterization of SIMO Wild-Type and Mutant Enhancer Activity in Zebrafish and Mouse Reporter Transgenics Wild-type and mutant versions of the SIMO element were cloned into appropriate transgenic reporter vectors by the method described in Ravi et al. The SIMO G>T mutation was PCR amplified from the subject’s DNA, and the artificial multinucleotide PAX6 BS mutation was made with Quickchange site-directed mutagenesis. Primers used are shown in Table S4. (A and B) Lateral views of transgenic mouse embryos with the wild-type element show expression in surface ectoderm at E9.5 (se) and lens at E10.5 dpc and in hindbrain (hb) and diencephalon (d). (C) At later developmental stages, X-gal staining is found in lens epithelium and neuroretina. (D–F) Comparable expression is seen in stable transgenic zebrafish with mouse SIMO element (mSIMO). (D) At 24 hr postfertilization (hpf), no GFP signal is yet detected. (E) By 48 hpf, strong enhancer activity is seen in the lens and more variably in diencephalon and hindbrain. (F) Expression is maintained at 72 hpf. (G–I) Double fluorescent reporter transgenic zebrafish demonstrate the loss of lens activity for the mutant SIMO elements. (G) Wild-type mouse SIMO linked to GFP (mSIMO-GFP) in green and the multinucleotide SIMO mutant linked to mCherry (mSIMO(Pax6)-CHR) in red. (H) Loss of enhancer activity in the eye by the patient mutation is demonstrated in comparative analysis of wild-type human hSIMO-G-GFP versus the mutant hSIMO-T-Cherry. (I) The same result is seen when the reporters are swapped. (J–R) Absence of eye expression in SIMO mutant transgenic embryos. (J and K) Two independent transgenic embryos for the multinucleotide SIMO mutant (mSIMO(Pax6)-Z) show absence of lens expression. (L) Staining is absent in SIMO mutant eyes at E17.5. (M) The human SIMO-G wild-type element is expressed in lens, diencephalon, and hindbrain. (N and O) Sections through E17.5 hSIMO eyes show staining in lens epithelium, neuroretina, and ciliary margin (arrowhead) in hSIMO-G (N) but not in hSIMO-T (O). (P–R) Three independent transgenic embryos for hSIMO-T at E10.5 demonstrate loss of expression in the lens while di- and rhombencephalon expression remains.

Figure 3

Pax6 Binds to the SIMO Enhancer and Is Essential for Its Function (A) Chromatin immunoprecipitation on chromatin, prepared from approximately 200 dissected mouse E14.5 embryonic eyes as described, shows that the SIMO element is enriched for enhancer-associated histone modifications H3K4me1 (ab8895, Abcam) and H3K27Ac (ab4729, Abcam; Table S3). The Pax6 P1 promoter is also positive for these marks, whereas featureless control fragments located near the adjacent Wt1 and Rcn1 genes show minimal enrichment. (B) ChIP with Pax6 antibody (DSHB) shows clear enrichment at the SIMO enhancer and Pax6 P1 promoter but not at the control regions. qPCR primer sets used are shown in Table S4. Relative enrichments are shown as mean percentage of input ± SEM (standard error of the mean); ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t test). (C) Double-stranded DNA affinity capture assay from nuclear extract prepared from approximately 100 E14.5 mouse embryonic eyes via NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, catalog number 78833), performed as described in Jeong et al. Pax6 is efficiently pulled down with a probe representing the wild-type SIMO-G sequence (Table S4). Binding to the mutant SIMO-T fragment is greatly reduced. Immunoblot via a mix of monoclonal anti-Pax6 antibodies (Table S3). Lane 1: precleared supernatant (Input); lane 2: bead wash fraction after extract binding; lane 3: final bead fraction via the wild-type SIMO-G probe; lane 4: final bead fraction for the mutant SIMO-T probe; lane M: marker. (D and E) Absence of functional Pax6 protein in vivo disrupts SIMO enhancer activity in the surface ectoderm and primordial lens of homozygous smalleye (Sey) embryos at E9.5 (D) and E10.5 (E). (F, H, and I [bottom embryo]) Injection of morpholinos (MO; Gene Tools) against zebrafish pax6.1a (5′-AGTTCCAACAGCCTTTGTATCCTCG-3′) and pax6.1b (5′-GCCTGAGCCCTTCCGAGCAAATCAG-3′) in stable SIMO-GFP transgenic fish embryos results in loss of reporter expression specifically in the lens, with variable phenotypic deformity of the embryos, notably a reduced eye size (arrows). (G, H, and I [top embryo]) Injection with the Gene Tools standard negative control morpholino: 5′-CCTCTTACCTCAGTTACAATTTATA-3′ has no effect. White arrowhead indicates the eye. Abbreviations are as follows: d, diencephalon; e, eye; hb, hindbrain; l, lens; nr, neuroretina.

Figure 4

The SIMO Enhancer Is Essential for Expression of Pax6 in Its Wider Genomic Context (A) Schematic representation of the modified BAC constructs containing the X. tropicalis Pax6 genomic locus, differing by the presence (orange ellipse) or absence (open triangle) of the SIMO element. Modified BACs are based on X. tropicalis BAC CH216-109E08 (obtained from CHORI BACPAC), transformed into the recombineering permissive bacterial strain SW102. A gfp3 reporter cassette was inserted into the pax6 exon 4 translational start site by GalK positive/negative selection. Tol2 recombinase arms were then placed in the BAC vector by the iTol2-ampicillin selection cassette. A 1.1 kb fragment covering the genomic coordinates from scaffold_399:567,254–568,353 (JGI 4.1/xenTro2), containing the X. tropicalis conserved SIMO sequence, was removed from the SIMO deleted BAC via rpsL/kanamycin selection recombineering. Primer sets used are shown in Table S4. The multitude of cis-elements present in the Pax6 locus is represented by blue ellipses, including the lens-specific ectodermal enhancer (EE, orange). (B–K) Temporal series of stable transgenic zebrafish carrying wild-type (B–F) or SIMO-deleted (G–K) reporter BAC transgenes. GFP signal recapitulates the combined pax6 expression pattern, including forebrain (fb), hindbrain (hb), spinal cord (sc), neuroretina (nr), lens (l), and pancreas (p). Wild-type and SIMO-deleted reporter patterns are identical at 24 hpf and lens expression is seen in both (B, C, G, H). By 48 hpf, GFP signal is absent from the lenses of SIMO-deleted BAC transgenic embryos, although being maintained in wild-type BAC transgenics (D, I). GFP patterns remain similar in wild-type and SIMO-deleted BAC transgenic fish at 72 hpf and 5 days with the exception of lens expression (E, F, J, K). (L) Model for the role of the autoregulatory PAX6 binding site in the SIMO enhancer, maintaining a positive feedback loop required for PAX6 expression during eye development. Undefined enhancer(s) in the locus, possibly including EE, initiate PAX6 transcription in early lens/surface ectoderm. Expression of PAX6 allows activation of the SIMO enhancer which, alone or in combination, maintains expression of PAX6 at subsequent stages of eye development.