The presence of about 6-7% of unknown mutations in CF patients (after the whole scanning of the coding region including macrodeletions) and the heterogeneous phenotype of the disease suggested the involvement of still unknown regulatory elements, which regulate the CFTR gene expression. Recently, it was found a relative paucity of sequence variation in the promoter region of CFTR gene but its relationship to CF remains yet uncertain (6).
In the present project, we will analyze the CFTR promoter region to evaluate the presence of allelic variants in two groups of CF patients either with “classical” or “atypical” form of the disease bearing undefined genotype. In particular, we want to clarify if promoter gene variants may represent causative-mutations either by rendering the regulatory regions unable to bind specific protein factors or by giving raise to novel binding sites for unpredicted transcription factors. The mutated CFTR promoter sequences will be transfected in suitable cell lines normally expressing the CFTR protein upstream a reporter gene in order to evaluate if the presence of mutations affect gene expression regulation.
The mutated sequences that will be related to an unbalance of the gene report expression will be selected for the functional proteomic experiments. In details the oligonucleotide sequence corresponding to a suitable mutated CFTR promoter sequence will be synthesised with a biotin tag and used as bait. The oligonucleotide will be immobilised on Streptavidin magnetic beads and incubated with a total/nuclear cell extract to isolate its interacting proteins by affinity purification (7). The same experiment will be carried out using the non mutated oligonucleotide as control. The proteins fished out by the baits will be separated by SDS-PAGE and identified by mass spectrometry methodologies and protein databases search. The identification of proteins normally involved in the binding of normal CFTR promoter will allow us to further elucidate the gene regulation mechanisms. Moreover, the comparison between the protein ligands specifically captured by the normal and the mutated promoter sequences will lead to the recognition of specific interactors involved in the disease and then to the correlation between genotype and CF phenotypes.