Table 1.
Synonymous and non-synonymous mutations identified through direct sequencing of 1126 cases with CHD and 1227 controls.
Figure 1.
(A) Schematic diagram of human CITED2 showing conserved regions (CR) 1–3 and the serine-glycine rich junction (SRJ). Also indicated are the locations of CITED2 variants. All unique non-synonymous variants found in this study are shown above the figure with variants found in the SRJ highlighted. Unique non-synonymous variants found by Sperling et al., 2005 (*) and Yang et al., 2010 (+) are shown below. SRJ comprises AA 161 to 199. (B) The CITED2 peptide sequence is shown for Homo sapiens (Human, HSCITED2, AF129290), CITED2-MRG1 (Human, HSCITED2 MRG1 isoform lacking the entire SRJ), Pan troglodytes (Chimapanzee, PTCITED2), Nomascus leucogenys (Gibbon, NLCITED2), Loxodonta africana (Elephant, LACITED2), Sus scrofa (Pig, SSCITED2), Bos taurus, (Cow, BTCITED2), Canis familiaris (Dog, CFCITED2), Mus musculus (Mouse, MMCITED2), Rattus norvegicus (Rat, RNCITED2), Monodelphis domestica (Opossum, MDCITED2), Gallus gallus (Chicken, GGCITED2), Anolis carolinensis (Anole lizard, ACCITED2), Xenopus laevis (African clawed frog, XLCITED2), Xenopus tropicalis (Western clawed frog, XTCITED2), and Danio rerio (Zebrafish, DRCITED2). Sequence alignments taken from the latest genome builds from EBI Ensembl. SRJ domain spans region marked with grey bar above corresponding to AA161 to 199 in the human protein sequence.
Figure 2.
CITED2 mutation and its functional consequences.
Hep3B cells were transiently transfected with the 3xAP2-luciferase reporter and the indicated plasmids and with CMV-lacZ. Results (mean±SEM, three independent experiments) are presented as relative luciferase units (RLU), corrected for β-galactosidase activity. The control transfection value (at the extreme left) in each case (with CMV-vector) is set at 1.
Figure 3.
Identification of residues in CITED2 wild type or CITED2T166N phosphorylated by ERK2 (MAPK1).
(A) GST-CITED2 wild type or mutant GST-CITED2T166N were incubated with Mg [32P]ATP in the presence of ERK2 and subjected to SDS-PAGE. The phosphorylated CITED2 was excised from the gel, digested with trypsin and the peptide separated by chromatography on a Vydac C18 column. The column was developed with an acetonitrile gradient (broken line) and 32P-radioactivity is shown (full line). The phosphopeptides A1 to A6 are indicated. (B) The major peaks (A1 to A6) of 32P-radioactivity were analysed by MALDI-TOF, MALDI TOF-TOF, Edman degradation and phosphoamino acid analysis as described in Materials and Methods and the data for A1–A3 is shown. The sequence inferred from this data is shown underneath each figure.
Figure 4.
MAPK signalling to CITED2 enhances its co-activation function.
Hep3B cells were transiently transfected with the 3xAP2-luciferase reporter, with CMV-lacZ, CITED2-expressing plasmid or the control vector and plasmids expressing MAPK1 or the control vector and with plasmids expressing a defective MAPKK1 (SV40-MAPKK1-S221A) or a constitutively active MAPKK1 (SV40-MAPKK1-S217ES221E). Results are presented as relative luciferase units corrected for lacZ activity. The control transfection value (at the extreme left) in each case (with CMV-vector) is set at 1.
Figure 5.
ES cell proliferation rescue by wild-type CITED2 and CITED2-T166N in the absence of leukemia inhibitory factor (LIF).
E14/T ES cells transfected by electroporation with the indicated plasmids were selected in puromycin. Surviving cells were plated in quadruplicate into 12-well plates in medium containing puromycin, but in the absence of LIF. The cells were fixed at the indicated time points using 10% formalin. The relative number of ES cells was determined by staining with 0.1% crystal violet, extracting cell-associated dye using 10% acetic acid, and measuring absorbance at A590. Results are presented as mean±SEM. Results represent a single experiment. Similar results were obtained from three independent experiments.
Figure 6.
(A) Targeting strategy for the generation of Cited2 T166N allele by homologous recombination. The T to N change was introduced by site directed mutagenesis into the orthologous residue in the mouse sequence. The structure of the wildtype Cited2 allele, targeting vector, targeted allele, and its structure after FLP mediated recombination are shown. The open reading frame (ORF, blue arrow) is entirely contained within exon 2 (exons indicated by grey rectangles). The targeting vector has an frt-PGK-NeoR-frt selection cassette downstream of exon 2, followed by a DTA (diphtheria toxin) cassette. (B) Targeting strategy for the generation of the Cited2attP ES cell line. A 5′ attP site was introduced into the first intron, upstream of the ATG and a 3′ attP site downstream of the stop codon and exon 2 as part of the neomycin selection cassette, in between the PGK promoter and the neomycin coding region. A DTA cassette was also introduced downstream of the 3′ homology arm for negative selection. (C) The human CITED2MRG1 isoform (not shown) and the human full length CITED2 were targeted into the Cited2 locus via PhiC31 integrase mediated cassette exchange. The exchange event occurs between the attP and attB sites giving rise to attL and attR sites. Successful exchange replaces the mouse Cited2 ORF with that of either the MRG1 isoform of CITED2 or the full length human CITED2 and brings the puromycin resistance gene under control of the PGK promoter.
Figure 7.
Phenotypic analysis of mouse embryos expressing CITED2 variants.
MRI analysis of embryos 15.5 days post coitum (dpc). Genotypes are indicated as shown. Sagittal sections through the left kidney (A–E) are shown to indicate the left adrenal gland where present (arrows), and absent (arrowhead). Transverse sections through the thorax (F–J) and 3D reconstructions (K–O) are shown to demonstrate cardiac anatomy. Loss of Cited2 leads to adrenal agenesis (B, arrowhead), right atrial isomerism, ventricular septal defect (VSD) and common atrium (G), and abnormal ventricular topology (L). Embryos expressing only the T166N variant, the MRG1 isoform, or full length human CITED2 have normal adrenal glands and hearts. RA, Right Atria; RV, Right Ventricle; LV, Left Ventricle; IVS, Interventricular Septum; IAS, Intra-atrial Septum; AAo, Aorta; AoA, Aortic Arch; Tr, Trachea; DAo, Dorsal Aorta; LSVS and RSVS, Left and Right Systemic Venous Sinus. Axis: A, Anterior; P, Posterior; V, Ventral; D, Dorsal; L, Left; R, Right. Scale bars: 0.5 mm.
Table 2.
Mice expressing a single copy of the Cited2 T166N (A) or Cited2 MRG1 (B) allele are found at the expected Mendelian ratios at weaning.
Figure 8.
Molecular characterization of the Cited2 T166N, Cited2 MRG1 and Cited2 HUM alleles.
(A) Western blot of total protein lysates from mouse embryonic fibroblasts (MEFs), probed with anti-CITED2 antibody. CITED2 and CITED2-MRG1 are indicated, as is a non-specific band (N.S.) that migrates at 25 kDa. (B) RT-PCR showing RNA products expressed by embryos of various genotypes. PCR primers were designed to differentiate between the endogenous mouse Cited2 transcript and the Cited2 MRG1 transcript by their size difference. Wild type mouse Cited2, containing the SRJ domain produces the larger 725 bp band. (C) Southern blots of Cited2 T166N allele. Top, Southern blot of EcoRI digested genomic DNA probed with a 5′-probe. Middle, Southern blot of BglII digested genomic DNA, probed with a 3′-probe. Probe positions are indicated in Fig. 3. Bottom, Southern blot of SpeI digested genomic DNA hybridized with an internal (Neomycin) probe, to confirm single copy integration. (D) Southern blots of Cited2 MRG1 and Cited2 HUM alleles. Top, Southern blot of EcoRI/SacII digested genomic DNA, probed with a 5′-probe. Middle, Southern blot of BglII digested genomic DNA, probed with a 3′-probe. The position of the probes is indicated in Fig. 3. Bottom, Southern blot of EcoRI digested genomic DNA hybridized with an internal (Puromycin) probe to confirm single copy integration.