Abstract
CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient.
Highlights
Congenital heart disease (CHD) is one of the major causes of childhood morbidity and mortality in the West
We found that CITED2-T166N is expressed at wild-type levels, binds EP300 and transcription factor AP2 (TFAP2) efficiently in vitro, and is localised to the nucleus excluding these factors as possible mechanisms for impaired function (Fig. S2)
Our resequencing study reported here and those reported elsewhere [16,17] indicate that non-synonymous mutations of CITED2 can be observed in patients with congenital heart disease, and that these mutations tend to cluster in the Serine-Glycine Rich Junction (SRJ) domain
Summary
Congenital heart disease (CHD) is one of the major causes of childhood morbidity and mortality in the West. Genetic variants have been shown to be associated with sporadic, non-Mendelian/ non-chromosomal CHD as non-synonymous disease-associated mutations have previously been found in case-control studies [5]. CITED2 is a CREBBP/EP300-interacting protein that is present in all vertebrates It is highly conserved in placental mammals, with 95% identity between human and mouse. It has three regions (CR1-3) that are conserved in other CITED family members, and an unusual Serine-glycine Rich Junction (SRJ, residues 161–199), which is unique to CITED2 [6,7,8,9,10]. The function of CR2 (residues 215–270) is to bind the CH1 domain of CREBBP and EP300 transcriptional co-activators, and in vitro studies indicate that it is necessary for all known biological activities of CITED2 [10,11,12,13]. The SRJ domain has been hypothesized to be a mutational hotspot as variants clustering in this region have previously been reported in patients with CHD [16,17]
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