Analisi dei siti di legame per AP-2α nella regione regolatoria di ESDN

The coordination of various biological functions as well as the response to environmental and developmental stimuli are governed by biochemical processes that regulate gene activity. Transcription is the initial step of gene expression and involves a multitude of transcription factors (TFs), their corresponding cis-acting elements on DNA, additional co-factors and the influence of chromatin structure. A number of functional genomic approaches have been developed to dissect the interplay between TFs and their cis-sequences, in order to identify TF regulatory targets, genome-wide, and to establish cellular transcriptional regulatory networks. A promising approach is to integrate microarray assays and in silico binding site (b.s.) predictions. The analysis of noncoding sequences of putative co-regulated genes is indeed useful in identifying common cis-regulatory elements recognized by specific TFs. The AP-2 transcription factor family consists of five related proteins (AP-2α, β, δ, γ, ε) sharing a common structure but encoded by different genes. AP-2 play relevant roles in a variety of cellular process, including cell growth, apoptosis, differentiation and migration, by binding to GC-rich sequences in the promoters of target genes and controlling their transcription. Many evidences show that AP-2 proteins are also involved in tumorigenesis, in particular in melanoma and mammary carcinomas, where they control the expression of a number of genes central to cancer development and progression. Comparison of gene expression between AP-2α-silenced (RNAi) and wild type HeLa cells was previously performed in our laboratory by whole-genome microarray analysis and a set of 721 (309 up-regulated and 412 down-regulated) differentially expressed genes was identified (Orso et al., 2008). The aim of this thesis work was to identify new direct transcriptional target genes of AP-2α by analyzing the regulatory regions of all the differentially expressed genes, integrating computational and experimental approaches. In addition we wanted to study, in detail, the AP-2α-driven regulation of the Endothelial Smooth muscle Derived Neuropilin like molecule (ESDN) gene, the most AP-2α regulated gene in the previous microarray analysis. The regulatory region (-900/+100, referring to the TSS as +1) of each AP-2α modulated gene was analyzed for the presence of AP-2α b.s., employing the canonical AP-2α Position Weight Matrix (PWM) reported in Jaspar database and at least one or two high score AP-2α b.s. was/were identified in many genes (363 with 1 b.s.; 264 with 2 b.s.). The direct binding of AP-2α to those sequences was investigated and confirmed for ten selected candidate genes, including ESDN, by Chromatin Immunoprecipitation (ChIP) assay. By performing a Gene Ontology analysis for the 264 genes containing at least 2 high score AP-2α b.s. a central role in cell motility and development was found for many genes. Three high and several low score AP-2α b.s. were identified in the promoter region of the ESDN gene by computational analysis. AP-2α-driven regulation of ESDN was examined by cloning the ESDN putative promoter region into a Luciferase reporter vector and by performing several reporter assays in tumor cells expressing different levels of AP-2α such as the MDA MB 231 and HeLa cell lines. To demonstrate the functional role of every single high score AP-2α b.s. and to assess their contribution to the overall ESDN promoter activity several deletion and site-directed mutant constructs were generated and their activity was measured and compared with the ESDN-wild type expression vector. AP-2α-driven regulation of the ESDN gene was demonstrated, and the contribution of each high score AP-2α b.s. evaluated in detail.