RESUMO
Two syndromic cognitive impairment disorders have very similar craniofacial dysmorphisms. One is caused by mutations of SATB2, a transcription regulator and the other by heterozygous mutations leading to premature stop codons in UPF3B, encoding a member of the nonsense-mediated mRNA decay complex. Here we demonstrate that the products of these two causative genes function in the same pathway. We show that the SATB2 nonsense mutation in our patient leads to a truncated protein that localizes to the nucleus, forms a dimer with wild-type SATB2 and interferes with its normal activity. This suggests that the SATB2 nonsense mutation has a dominant negative effect. The patient's leukocytes had significantly decreased UPF3B mRNA compared to controls. This effect was replicated both in vitro, where siRNA knockdown of SATB2 in HEK293 cells resulted in decreased UPF3B expression, and in vivo, where embryonic tissue of Satb2 knockout mice showed significantly decreased Upf3b expression. Furthermore, chromatin immunoprecipitation demonstrates that SATB2 binds to the UPF3B promoter, and a luciferase reporter assay confirmed that SATB2 expression significantly activates gene transcription using the UPF3B promoter. These findings indicate that SATB2 activates UPF3B expression through binding to its promoter. This study emphasizes the value of recognizing disorders with similar clinical phenotypes to explore underlying mechanisms of genetic interaction.
Assuntos
Transtornos Cognitivos/genética , Anormalidades Craniofaciais/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Knockout , Fenótipo , Regiões Promotoras Genéticas , Síndrome , Fatores de Transcrição/genéticaRESUMO
Satb1 and Satb2 have been recently described as regulators of embryonic stem (ES) cell pluripotency and as silencing factors in X chromosome inactivation. The influence of the pluripotency machinery on X chromosome inactivation and the lack of an X chromosome inactivation defect in Satb1(-/-) and Satb2(-/-) mice raise the question of whether or not Satb proteins are directly and/or redundantly involved in this process. Here, we analyzed X chromosome inactivation in fibroblastic cells that were derived from female Satb1(-/-)Satb2(-/-) embryos. By fluorescence in situ hybridization to visualize Xist RNA and by immunohistochemistry to detect H3K27me3 histone modifications, we found that female Satb1(-/-)Satb2(-/-) fibroblastic cells contain proper Barr bodies. Moreover, we did not detect an upregulation of X-linked genes, suggesting that Satb proteins are dispensable for X chromosome inactivation in mice.
