MOZ regulates the Tbx1 locus, and Moz mutation partially phenocopies DiGeorge syndrome

Abstract

DiGeorge syndrome, caused by a 22q11 microdeletion or mutation of the TBX1 gene, varies in severity greatly, even among monozygotic twins. Epigenetic phenomena have been invoked to explain phenotypic differences in individuals of identical genetic composition, although specific chromatin modifications relevant to DiGeorge syndrome are elusive. Here we show that lack of the histone acetyltransferase MOZ (MYST3/KAT6A) phenocopies DiGeorge syndrome, and the MOZ complex occupies the Tbx1 locus, promoting its expression and histone 3 lysine 9 acetylation. Importantly, DiGeorge syndrome-like anomalies are present in mice with homozygous mutation of Moz and in heterozygous Moz mutants when combined with Tbx1 haploinsufficiency or oversupply of retinoic acid. Conversely, a Tbx1 transgene rescues the heart phenotype in Moz mutants. Our data reveal a molecular mechanism for a specific chromatin modification of the Tbx1 locus intersecting with an environmental determinant, modeling variability in DiGeorge syndrome.

Figure 1

Craniofacial, Palate, Aortic Arch, and Heart Abnormalities in Moz^Δ/Δ Mutant Pups Moz^+/+ (A, C, E, G, I, K, N, S, U, and W) and Moz^Δ/Δ (B, D, F, H, J, L, M, O, T, V, and X) newborn mouse pups (A–F), E18.5 pups (G–O), E9.5 (S and T), and E10.5 (U–X) embryos. Lateral view of the head (A and B), ventral view of the palate (C and D), and of the bone structure of the palate (E and F). Note the shortened face and mandible of the mutant (A and B), the lack of midline fusion of the secondary palate (arrow in D and F). Asterisks in (F) indicate location where the maxillary shelf and the palatine shelf should have fused. Aortic arch and other great vessels, ventral view, ink injected (G and H), and traced (I and J). Note the absence of the aortic arch between the LCCA and the LSA, i.e., an IAA-B (arrow in H and J) and the connection of the pulmonary trunk with the descending aorta (H and J). Resin cast of heart and large blood vessels (K–M) injected with red (left ventricle) and blue (right ventricle), H&E sections of hearts, ventral view (N and O). Note the complete filling of both sides of the Moz^Δ/Δ hearts by injection from either the right (L) or left (M) ventricle, the ventricular septum defect (arrow in O), and overriding aorta (asterisk in O). Schematic drawing of aortic arch development from E11.5 to E18.5 (P and Q) and abnormal vessels in E18.5 Moz^Δ/Δ mutants (R). Ink-injected aorta-pharyngeal complex at E9.5 (S and T) and E10.5 (U and V). Resin cast of the aorta-pharyngeal complex at E10.5 (W and X). The connection between the ascending and descending aspects of the aorta is derived from the left fourth pharyngeal artery (red in P and Q), missing in Moz^Δ/Δ (arrow, R). The left fourth pharyngeal artery is not yet developed at E9.5 in either Moz^+/+ or Moz^Δ/Δ (S and T). Note, however, the presence of a fourth PAA in Moz^+/+ E10.5 embryos (U and W), which is absent in Moz^Δ/Δ E10.5 embryos (4′ in V and X). 1b, mandibular portion of the first pharyngeal arch; 2, second pharyngeal arch; 3, 4, 6, third, fourth, and sixth PAA; arrowhead in (F), presphenoid bone, only visible through cleft palate; Ao, aorta; As, alisphenoid; Bs, basisphenoid; dAo, dorsal aorta; Ca, caudal; DA, ductus arteriosus; He, heart; LA, left atrium; LCCA, left common carotid artery; LSA, left subclavian artery; LPA, left pulmonary artery; LV, left ventricle; M, maxillary shelf; Mx, maxilla; OV, otic vesicle; P, palatine shelf; P2, secondary palate; Pa, pulmonary artery; Pm, premaxilla; PP, primary palate; PT, pulmonary trunk; RA, right atrium; RCCA, right common carotid artery; Ro, rostral; RSA, right subclavian artery; RV, right ventricle; SA, subclavian artery; vAo, ventral aorta; VS, ventricular septum. Z, zygomatic process. Scale bar, 5 mm (A and B), 2 mm (C and D), 1.5 mm (E and F), 940 μm (G and H), 2.3 mm (K), 1.5 mm (L), 1.2 mm (M), 550 μm (N and O), 1150 μm (S and T), 290 μm (U and V), and 230 μm (W and X). n = 33 Moz^Δ/Δ, 45 Moz^Δ/+, and 35 Moz^+/+ newborn pups (A–F), n = 19 Moz^Δ/Δ,13 Moz^Δ/+, and 15 Moz^+/+ newborn pups (G–J), n = 19 Moz^Δ/Δ, 16 Moz^Δ/+, and 17 Moz^+/+ newborn pups (K–O). n = 12 Moz^Δ/Δ, 14 Moz^Δ/+, and 9 Moz^+/+ E9.5 embryos (S and T), n = 4 Moz^Δ/Δ, 20 Moz^Δ/+, and 13 Moz^+/+ control E10.5 embryos (U–X). See also Figure S1.

Figure 2

mRNA Expression Levels of Genes Regulating Heart Development and Genes within the 22q11 Deletion Interval (A) Schematic drawing of the genomic context of the Tbx1 gene and genes flanking the locus on chromosome 16 in the mouse, a region syntenic with the 22q11 deletion interval in human. (B) Schematic representation of major regulators of transcription during heart development. (C–I) mRNA expression levels as assessed by RT-qPCR and normalized to housekeeping genes in Moz^+/+ and Moz^−/− embryos at E8.5 (C and D), E9.5 (E–H), and E10.5 (I); mRNAs encoded by genes as indicated in the graphs. ^∗p < 0.05 and ^∗∗p < 0.01. Exact p values are given below. Significantly lower in Moz^−/− embryos were mRNAs encoded by Tbx1 at E8.5 (C, p = 0.029), at E9.5 (E, p = 0.001), and E10.5 (I, p = 0.039); Tbx5 at E9.5 (E, p = 0.011) and E10.5 (I, p = 0.006) and Tbx2 at E10.5 (I, p = 0.014). Not significantly changed were the mRNA expression levels of housekeeping genes (D and H), other genes in the 22q11 deletion interval (F), and major regulators of heart development, Gata4 and Nkx2-5 (G). Data are presented and were analyzed as described in the Experimental Procedures. Data were derived from 36 embryos, n = 6 Moz^+/+ and 6 Moz^−/− per gene at each developmental stage.

Figure 3

Reduced Expression of Tbx Genes in Moz^Δ/Δ Embryos Detected by In Situ Hybridization Moz^+/+ (A, C, E, G, and I) and Moz^Δ/Δ (B, D, F, H, and J) embryos, whole mount at E10.5 (A–D and G–J), and sections at E9.5 (E and F). Expression of Tbx1 (A–F), Tbx2 (G and H), and Tbx5 (I and J). Note the substantial reduction (B) or more moderate reduction (D and F) in Tbx1 mRNA signal in the Moz^Δ/Δ embryos (dark purple in whole mount), reduced Tbx1 mRNA signal in the mesodermal core of the pharyngeal arches (black-silver grains, arrows E and F), and in the pharyngeal endoderm (asterisks, E and F) in the Moz^Δ/Δ embryos on sections. The region of the fourth pharyngeal arch is indicated (arrows, C and D). Note the reduction in Tbx2 (G and H) and Tbx5 mRNA signals (I and J, arrowhead indicates Tbx5 expression maintained in the inflow tract at E10.5). 1a and 1b, maxillary and mandibular, respectively, aspect of the first pharyngeal arch; 2, 3, 4, second, third, and fourth pharyngeal arch; 4′, region where the fourth pharyngeal arch should be present in the Moz^Δ/Δ; FL, forelimb bud; He; Heart; OV, otic vesicle. Scale bar, 190 μm (A and B), 390 μm (C and D), 142 μm (E and F), 350 μm (G and H), and 530 μm (I and J). Data were derived from 42 embryos, n = 6 Moz^+/+ and 6 Moz^Δ/Δ per gene at E10.5 and 3 Moz^+/+ and 3 Moz^Δ/Δ at E9.5. See also Figure S2.

Figure 4

H3K9ac and MOZ Complex Protein ING5 Occupancy Is Reduced at the Tbx1 and Tbx5 Gene Loci in the Absence of MOZ (A) Schematic drawing of the genomic loci assayed for histone modifications and ING5 occupancy by ChIP. “Tbx1_1, Tbx1_2, Tbx2_1 etc.” denote sequences amplified in (B)–(K). H3K9ac (B–D), H3K14ac (E–G), H3K9me3 (H and I), and ING5 (J and K) were assessed by ChIP-qPCR of Moz^−/− and Moz^+/+ E10.5 embryos for sequences −0.7 to −1.0 kb upstream of the TSS (B, E, H, and J; tiles_1), in proximity of the transcription start site (TSS; C, F, H, J, and K; tiles_2) and in the coding region (D and G). Note the reduction in H3K9ac in Moz^−/− mutants −0.7 to −1 kb upstream of the TSS of Tbx1, Tbx2, and Tbx5 (B; p < 0.0005, p = 0.003, and p = 0.003, respectively), near the TSS of Tbx1 and Tbx5 (C; p = 0.032 and p = 0.002, respectively), and in the Tbx1 coding region (D; p = 0.0151), but no reduction in H3K14ac (E–G) and an increase in H3K9me3 (H and I; p = 0.0071). Notice the reduction in binding of the MOZ complex protein ING5 to Tbx1 and Tbx5 in Moz^−/− mutants (J; p = 0.001 and p < 0.0005, respectively), but not Tbx2 and Tbx3 (K). For the effects of Moz mutation, p values <0.05, <0.01, and <0.001 are indicated by one, two, and three asterisks, respectively. Data are presented and were analyzed as described in the Experimental Procedures. Data are derived from a total of 39 embryos, n = 6 Moz^+/+ and 6 Moz^−/− per ChIP antibody (B–G), 3 Moz^+/+ and 3 Moz^−/− (H, I, and K), and 4 Moz^+/+ and 5 Moz^−/− (J).

Figure 5

Expression of Genes Encoding Regulators of the Tbx1 Gene, Genes Regulated by TBX1 Protein, and Genes Showing Genetic Interaction with the Tbx1 Locus mRNA levels of genes as indicated assessed by RT-qPCR (A–E) and occupancy of gene loci as indicated by FOXA2 (F) and by the MOZ complex protein ING5 (G). Expression of the genes encoding the forkhead protein FOXA2 (A–C) reported to upregulate Tbx1 gene expression, as well as the Chd7 gene (E), reported to interact genetically with the Tbx1 locus, is unaffected by Moz mutation at E8.5 (A), E9.5 (B and E), and E10.5 (C). Tbx1 locus occupancy assessed by ChIP-qPCR by FOXA2 is increased in E9.5 Moz^−/− mutants (F; p = 0.015), albeit without being able to induce normal Tbx1 expression levels in the absence of MOZ (Figure 2). Expression of Foxc1 is increased at E8.5 (A, p = 0.04), and expression of Foxc1 and Foxc2 is decreased at E9.5 (B, p = 0.008 and p = 0.001, respectively) and E10.5 (C, p = 0.034 and p = 0.011, respectively) in Moz^−/− mutants without changes in occupancy of Foxc1 locus by ING5 (G; p = 0.251) and without occupancy of the Foxc2 locus by ING5 (data not shown). The expression of the Pitx2 gene, a target gene of TBX1, is decreased at E9.5 and E10.5 in Moz^−/− mutants (D, p = 0.023) without changes in ING5 occupancy of the Pitx2 locus (G). For the effects of Moz mutation, p values <0.05 and <0.01 are indicated by one and two asterisks, respectively. Data are presented and were analyzed as described in the Experimental Procedures. Data were derived from 48 embryos, n = 6 Moz^+/+ and 6 Moz^Δ/Δ per developmental stage (A–E) and 3 Moz^+/+ and 3 Moz^−/− (F and G).

Figure 6

Genetic Synergy between Moz and Tbx1 Haploinsufficiency and Rescue of Moz Mutant Heart Defects by a Tbx1 Transgene (A) Survival of wild-type, Moz^+/− and Tbx1^+/− single-heterozygous, and Moz^+/−Tbx1^+/− double-heterozygous mice to weaning. Note that 80% of Moz^+/−Tbx1^+/− double-heterozygous mice are not viable. (B) Incidence of DGS-like anomalies observed in Moz^+/−Tbx1^+/− double- and single-heterozygous mice at E17.5–E18.5. (C and D) Details of DGS-like anomalies in locations other than the heart (C) and in the heart (D) observed in the Moz^+/−Tbx1^+/− double and single heterozygotes. (E and F) E18.5 offspring in three litters of Moz^Δ/+Tbx1-Tg by Moz^Δ/+ matings. (E) Overview and high-resolution images of VSDs in three Moz^Δ/Δ animals (arrows, as in Figure 1 and Table 1) and the rescue of the septal defects by one copy of the Tbx1-containing BAC RP23-35B9 in three Moz^Δ/ΔTbx1-Tg animals (serial images in Figure S3). (F) Summary of all 25 offspring. The septal defects observed in the four Moz^Δ/Δ were three VSDs and one aortopulmonary septal defect. Data are presented and analyzed as described in the Experimental Procedures. AbRSA, abnormal right subclavian artery including retrotracheal and abnormal origin; GVRD, great vessel wall remodeling defects, i.e., dilated in diameter and thin walled; LA, left atrium; LV, left ventricle; IAA-B, interrupted aortic arch type B; RA, right atrium; RV, right ventricle; VS, ventricular septum; VSD, ventricular septal defect. Scale bar, 445 μm in the left two panels of (E) and 185 μm in all other panels of (E).

Figure 7

Sensitization to RA by Loss of One Copy of Moz (A) Effects of RA treatment at E7.25–E9.25 on Moz^+/+, Moz^Δ/+, and Moz^Δ/Δ E17.5–E18.5 pups on the incidence of DGS-like anomalies. Note the induction of DGS-like anomalies in heterozygotes treated with RA at E8.25 and E9.25. (B) Details of anomalies observed in the RA-treated Moz^Δ/+ heterozygous animals. Data are presented and analyzed as described in the Experimental Procedures. Abnormal RSA, abnormal right subclavian artery including retrotracheal and abnormal origin; IAA type B, interrupted aortic arch type B; TGA, transposition of the great arteries.