Caracterización funcional y estructural de las secuencias en cis y factores en trans presentes en la región LCR del gen de la tirosinasa de ratón

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-03-2005Tyrosinase, the ratdimiting enzyrne in the melanin synthesis pathway, constitutes an interesting model to study the mechanisrns by which expression domains work. The expression studies on the mouse tyrosinase gene regulation are an example of a representative mammalian expresslon domain, regarding ik organization within the nuclear chromatin, the establishment of its specific pattem of expression during development and throughout the life of an organlsm. Recently, functional dissection of regulatory elements present within this LCR have revealed the existente of different regulatory activities, including a novel boundary eiement. In this report, we have studied the relevant cis sequences, involved in the insulation of the tyrosinase gene and the hns acting factors binding to #ose within the mouse tyrosinase LCR. We have found several DNA-protein complexes binding to the LCR. We have identified one of them, binding to a G rich sequence, the CTCF factor, which has been previously described as an insulator protein. Taking advantage of the complexion of the human genome, and the comparative analysis, we have found a new 5'upstream regulatory sequence in the human tyrosinase gene, by comparison of the mouse LCR, which is active functionally. On the other hand, we have ohserved the chromatin status for the LCR region, founding a methylation and acetylation pattern which suggest a transition between open and close status of the chromatin within tyrosinase domain. Finally, we have generated two new genomic tools to further investigate the role of the LCR region at the endogenous location. ñrst, a v e r designed for homologous recombination in ES cell, and cecond, a transgene expressing the inducible Cre recombinase under the control of tyrosinase regulatory regions