Comparative genomic sequence analysis coupled to chromatin immunoprecipitation: a screening procedure applied to search for regulatory elements at the RET locus.
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.
Puppo
Francesca
F
Laboratory of Molecular Genetics, Giannina Gaslini Institute, Genova, Italy.
Musso
Marco
M
Pirulli
Doroti
D
Griseri
Paola
P
Bachetti
Tiziana
T
Crovella
Sergio
S
Patrone
Giovanna
G
Ceccherini
Isabella
I
Ravazzolo
Roberto
R
D000107:Acetylation; D000818:Animals; D045744:Cell Line, Tumor; D017931:DNA Primers; D015972:Gene Expression Regulation, Neoplastic; D006627:Hirschsprung Disease / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D009447:Neuroblastoma; D016133:Polymerase Chain Reaction; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D051381:Rats; D013964:Thyroid Neoplasms; D014158:Transcription, Genetic
D017931:DNA Primers; D006657:Histones; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C494999:Ret protein, mouse