The molecular role of GTF2IRD1: a protein involved in the neurodevelopmental abnormalities of Williams-Beuren syndrome

Abstract

Background: GTF2IRD1 is a member of the GTF2I gene family, located on chromosome 7 in a region prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. Human mapping data and analyses of mouse knockouts implicate GTF2IRD1 as the prime candidate for the craniofacial abnormalities, mental retardation, visuospatial construction deficits and hypersociability of WBS. Aims: The aim of this work was to study the cellular and molecular role of GTF2IRD1 by investigating: i) the cellular localisation of GTF2IRD1; ii) its protein interacting partners; iii) the gene dysregulation caused by GTF2IRD1 loss; and iv) the presence of GTF2IRD1 in epigenetic complexes regulating gene expression. Results: i) Immunofluorescence analyses in mammalian cell lines and in human ES cell-derived neurons showed endogenous GTF2IRD1 as a nuclear speckle protein. The comparison of this punctate pattern with markers of nuclear sub-compartments and chromatin marks supports an association with developmentally regulated silent chromatin. ii) To define functional relationships, yeast two-hybrid screenings were used to isolate novel interaction partners. Most of the nuclear-localised interactions were validated in mammalian cells, being predominantly proteins involved in chromatin modification and transcriptional regulation. The sites of interaction in GTF2IRD1 were mapped to specific domains. iii) To identify transcriptional changes arising from GTF2IRD1 loss, microarray studies were conducted in siRNA-treated HeLa cells and brain tissue from Gtf2ird1 knockout mice. In the corpus striatum, qPCR validation indicated up-regulation of genes involved in neuronal development and immediate-early response genes that may explain some of the observed neurobehavioural phenotypes. iv) GTF2IRD1 was found to be involved in chromatin modifying complexes by direct associations with histone deacetylases and can affect their enzymatic activity. Conclusions: The results of this thesis indicate that GTF2IRD1 forms complexes with DNA-binding and chromatin modifying proteins to regulate gene expression through epigenetic mechanisms that are controlled in a tissue specific manner. The sites of protein interactions indicate key features regarding the evolution of GTF2IRD1 and integration with tight post-translational regulation, fitting well with the concept of human disease states caused by copy number variation