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Comparative genomic sequence analysis coupled to chromatin immunoprecipitation: a screening procedure applied to search for regulatory elements at the RET locus

¹ Laboratory of Molecular Genetics, Giannina Gaslini Institute, Genova
² Department of Pediatrics and Center of Excellence for Biomedical Research, University of Genova
³ Department of Genetics, Institute Burlo Garofolo, Trieste, Italy

RET gene expression is characterized by high tissue and stage specificity during the development of neural crest derivatives and in the pathogenesis of inherited cancer syndromes and Hirschsprung disease. Identifying all elements contributing to its transcriptional regulation might provide new clues to clarify both developmental and pathogenic mechanisms. We previously demonstrated that chromatin acetylation affects RET transcription; therefore, we have set up a strategy based on analysis of sequences conserved among species at the RET locus, combined with the characterization of their chromatin structure, to identify new potential regulatory elements. The histone acetylation level was evaluated by the chromatin immunoprecipitation method applied to cells displaying different degrees of endogenous RET expression. Real-time quantitative PCR of immunoprecipitated DNA-protein complexes and transfection experiments, with constructs expressing a reporter gene in which the putative regulatory regions are inserted, indicate a correlation between histone acetylation and endogenous RET expression and highlight conserved sequences with potential regulatory roles. This paper presents a reliable screening procedure to unearth elements able to affect gene regulation at the transcriptional level in a large genomic region.

Hirschsprung disease; comparative sequence analysis; histone acetylation; real-time quantitative PCR; transcriptional regulation

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