Identification of the TFII-I family target genes in the vertebrate genome

Abstract

GTF2I and GTF2IRD1 encode members of the TFII-I transcription factor family and are prime candidates in the Williams syndrome, a complex neurodevelopmental disorder. Our previous expression microarray studies implicated TFII-I proteins in the regulation of a number of genes critical in various aspects of cell physiology. Here, we combined bioinformatics and microarray results to identify TFII-I downstream targets in the vertebrate genome. These results were validated by chromatin immunoprecipitation and siRNA analysis. The collected evidence revealed the complexity of TFII-I-mediated processes that involve distinct regulatory networks. Altogether, these results lead to a better understanding of specific molecular events, some of which may be responsible for the Williams syndrome phenotype.

Fig. 1.

Pathway involvement of BEN- (A) and TFII-I- (B) modulated genes. y axis, P values for significance of enrichment; x axis, KEGG pathway terms, listed as follows: 1, arachidonic acid metabolism; 2, nitrogen metabolism; 3, hedgehog signaling pathway; 4, type I diabetes mellitus; 5, calcium signaling pathway; 6, maturity-onset diabetes of the young; 7, cell adhesion molecules (CAMS); 8, hematopoietic cell lineage; 9, cytokine–cytokine receptor interaction; 10, complement and coagulation cascades; 11, neuroactive ligand–receptor interaction; 12, cell cycle; 13, ribosome; 14, proteasome; 15, focal adhesion; 16, apoptosis; 17, insulin signaling pathway; 18, antigen processing and presentation; 19, adherens junction; 20, MAPK signaling pathway; 21, ATP synthesis; 22, gap junction; 23, citrate cycle (tricarboxylic acid); 24, oxidative phosphorylation; 25, TGFβ signaling pathway; 26, reductive carboxylate cycle (CO₂ fixation); 27, neurodegenerative disorders; 28, FcεRI signaling pathway; 29, Toll-like receptor signaling pathway; 30, dorso–ventral axis formation; 31, leukocyte transendothelial migration; 32, cysteine metabolism.

Fig. 2.

Alignment and classification of target genes. Venn diagram shows the alignment of microarray-modulated genes and bioinformatics promoter search results.

Fig. 3.

Classification of target genes with the TFII-I-binding motif. Candidate genes selected for further validation are shown in bold.

Fig. 4.

In vivo recruitment of BEN and TFII-I to proximal promoters of target genes. (A) ChIP analysis of target genes in MEFs endogenously expressing BEN and TFII-I. (B) Conservation of upstream elements containing the putative TFII-I-binding site. Fish species are shown for those genes where reliable alignments of 5′-untranslated regions are available. Identical nucleotides are starred. Thirty vertebrate sequences shown were used for calculation of logo with WebLogo software (30). The height of letters correlates with degree of conservation.

Fig. 5.

Validation of target genes by double siRNA knockdown of BEN and TFII-I in MEFs. Expression fold changes were calculated relative to control negative siRNA treatment for each gene. Data were analyzed by comparative Ct method using β-actin as a control.