Abstract
In mammalian organisms liquid tumors such as acute myeloid leukemia (AML) are related to spontaneous chromosomal translocations ensuing in gene fusions. We previously developed a system named bridge-induced translocation (BIT) that allows linking together two different chromosomes exploiting the strong endogenous homologous recombination system of the yeast Saccharomyces cerevisiae. The BIT system generates a heterogeneous population of cells with different aneuploidies and severe aberrant phenotypes reminiscent of a cancerogenic transformation. In this work, thanks to a complex pop-out methodology of the marker used for the selection of translocants, we succeeded by BIT technology to precisely reproduce in yeast the peculiar chromosome translocation that has been associated with AML, characterized by the fusion between the human genes NUP98 and TOP2B. To shed light on the origin of the DNA fragility within NUP98, an extensive analysis of the curvature, bending, thermostability, and B-Z transition aptitude of the breakpoint region of NUP98 and of its yeast ortholog NUP145 has been performed. On this basis, a DNA cassette carrying homologous tails to the two genes was amplified by PCR and allowed the targeted fusion between NUP145 and TOP2, leading to reproduce the chimeric transcript in a diploid strain of S. cerevisiae. The resulting translocated yeast obtained through BIT appears characterized by abnormal spherical bodies of nearly 500 nm of diameter, absence of external membrane and defined cytoplasmic localization. Since Nup98 is a well-known regulator of the post-transcriptional modification of P53 target genes, and P53 mutations are occasionally reported in AML, this translocant yeast strain can be used as a model to test the constitutive expression of human P53. Although the abnormal phenotype of the translocant yeast was never rescued by its expression, an exogenous P53 was recognized to confer increased vitality to the translocants, in spite of its usual and well-documented toxicity to wild-type yeast strains. These results obtained in yeast could provide new grounds for the interpretation of past observations made in leukemic patients indicating a possible involvement of P53 in cell transformation toward AML.
Highlights
Nucleoporins have important roles in many cellular pathways such as the nucleocytoplasmic transport [1], mitotic spindle assembly checkpoint [2], and chromatin metabolism [3]
We found that the identity (BLASTp) between HsNup98 and ScNup145 is on average 36% with peaks of 72% within short regions while the identity between HsTop2B and ScTop2 is on average much higher (Figure 1A) since topoisomerases are well conserved, with structural insights from the yeast enzymes that are likely to apply to the human ones [45]
Our data suggest that Nup98 could be related to P-bodies regulation in yeast and, be responsible for mRNA turnover in the cytoplasm
Summary
Nucleoporins have important roles in many cellular pathways such as the nucleocytoplasmic transport [1], mitotic spindle assembly checkpoint [2], and chromatin metabolism [3]. Among all the different possible chimeras leading to hematopoietic malignancies, we focused on a peptide resulting from the fusion between the genes NUP98 and TOP2B that had been found in a patient with primary AML [13]. An important point is that at least two different reciprocal chimeric transcripts (TOP2B–NUP98) were identified, suggesting that a precise reciprocal construct is not essential for the leukemic transformation process This observation is confirmed by other analyses of NUP98 fusions, where the reciprocal transcript was never found in leukemic patients [15, 16]. Since BIT produces always non-reciprocal chromosome translocations [17, 18], the demonstration that the reciprocal transcript(s) did not play a role in the oncogenic
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