i) Isolation of PML binding sites; ii) Isolation of PML target genes; iii) Isolation of PML/RARalpha and RARalpha target genes; iv) Isolation of PML binding proteins; v) Biophysical characterization of PML protein; vi) Generation of PML/RARalpha transgenic mice; vii) Generation of hemopoietic cell lines overexpressing PML, PML/RARalpha and RARalpha; viii) Characterization of HOX gene expression in myeloid cells; ix) Generation of hemopoietic cell lines overexpressing HOX genes.
i) Identification of CD2, Myc and ICAM-I genes as PML/RARalpha target genes; ii) Identification of one PML binding protein; iii) NMR structure of the PML protein; iv) Transgenization of MT-driven PML/RARalpha cDNA; v) Expression of PML/RARalpha into hemopoietic precursor cell lines and definition of its effect on differentiation and survival; vi) Regulation of HOXD4 gene by RA.
MAJOR SCIENTIFIC BREAKTHROUGHS:
Acute promyelocytic leukemia (APL) is characterized by the accumulation of blasts blocked at the promyelocytic stage of differentiation. The differentiation block is a major determinant in the maintenance of the leukemic phenotype, since induction of terminal differentiation with retinoic acid therapy leads to clinical remission. At the molecular level, most APL cases are characterized by the presence of the PML/RARalpha fusion gene, generated by the 15;17 translocation. The 15;17 translocation breakpoints were isolated by 3 of the 4 participants to this research network. The chromosome 17 breakpoint is located within the RARalpha locus, the chromosome 15 breakpoint within a previously unknown locus, named PML. As a consequence of the translocation a PML/RARalpha fusion gene is formed that encodes a PML/RARalpha fusion protein. The following are the major scientific achievements of the participants to this research network.
PML encodes a protein of unknown function localized in recently discovered nuclear structures (nuclear bodies) and contains a number of novel motifs including three Zn2+ binding domain and a alpha-helical coiled-coil domain with dimerization properties. Biophysical studies including CD and optical spectroscopy showed that the PML RING finger requires Zn2+ for autonomous folding and that cysteines are used in metal ligation. RARalpha is a RA-dependent enhancer factor that dimerizes with the RXR cofactor to directly bind to the promoter region of RA-target genes.
The PML/RARalpha fusion protein retains all the PML and RARa functional domains. Expression of the PML/RARalpha protein in the monocytic precursor leukemic cell line U937 blocks vitamin D3-induced differentiation and increases RA-induced differentiation. These biological effects of the PML/RARalpha protein might be responsible for crucial features of the APL phenotype, e.g. the accumulation of hematopoietic precursors blocked at the promyelocytic stage of differentiation and the high sensitivity to RA. The leukemogenetic potential of the PML/RARalpha fusion protein has been investigated using chicken bone marrow and is currently being investigated in mice expressing the PML/RARalpha protein (transgenic mice with PML/RARalpha cDNAs under the control, of inducible promoters).
Little is known on the mechanisms of the PML/RARalpha action. PML/RARalpha heterodimerizes with PML, forms homodimers which bind to RA-target promoters and heterodimers with RXR. Some of the putative PML/RARalpha regulated genes have been recently identified (CD2, myc, ICAM1).
Finally, the role of HOX genes during the hematopoietic development and their putative role as PML/RARalpha target genes is being investigated.
Funding SchemeCSC - Cost-sharing contracts
WC2A 3PX London