The unique biological features of the marine product Yondelis (ET-743, trabectedin) are shared by its analog ET-637, which lacks the C ring.

It was previously suggested that the peculiar mechanism of action of the novel anticancer drug Yondelis (ET-743, trabectedin) was due to part of the molecule, units A and B, binding to DNA in the minor groove, causing an alkylation at the N2 of guanine, while unit C protrudes out of DNA, possibly interacting with transcription factors or other DNA binding proteins. To test this hypothesis, we have compared the biological activity and the mode of action of Yondelis with its analogue ET-637, which has the same chemical structure except for the lack of the C ring. Yondelis and ET-637 showed similar cytotoxic potency and cell cycle perturbations. As already reported for Yondelis, the UV-96 cell line, deficient in ERCC-1, was less sensitive to ET-637 than the parental cell line. The binding of Yondelis or ET-637 to DNA-oligonucleotides was demonstrated by gel shift assay and SDS did not reverse the binding. Both compounds blocked the temperature-induced activation of the HSP40 promoter in the range of 1-10 nM. This study indicates that ET-637 acts similarly to Yondelis and demonstrates that the C ring of Yondelis may not be required for its biological activity.

NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices.

NF-Y (CBF) is a CCAAT-binding trimer that activates 25 % of eukaryotic promoters. It contains putative histone fold motifs (HFMs) and distorts DNA. By using electrophoretic mobility shift assays with the twin CCAAT boxes of the human gamma-globin promoter and several combinations of subunit mutants, we dissected some of the structural features of CCAAT-box binding. NF-YA and NF-YC Q-rich domains significantly influence bending angles quantitatively, but not qualitatively, since they do not modify DNA orientation. They are both required for co-operative interactions among NF-Y molecules: for this, a precise alignement of two CCAAT boxes, 32 bp, three turns of the helix, is essential. Unlike the wild-type (wt) protein, steric hindrance does not impede simultaneous binding of the mutant composed of the short homology domains to CCAAT boxes closer than 22 bp: the addition of 11 amino acid residues to NF-YB and 13 to NF-YC flanking the HFM, restores wt behaviour. These stretches are predicted to form H2B-like alphaC and H2A-like alphaN fourth helices. A further support to this hypothesis comes from off-rates analysis of mutant combinations: the half-life of NF-Y, which is dependent on the type of NF-YB used, is extremely shortened, when the putative alphaC is present, nearly as much as in the wt NF-YB. These data (i) provide further evidence that NF-YB-NF-YC belong to the H2B-H2A subclasses, (ii) uncover new features of Q-rich domains, and (iii) define rules for NF-Y synergy that are potentially important for the regulation of many eukaryotic promoters.

Conservation and divergence of NF-Y transcriptional activation function.

The CCAAT-binding protein NF-Y is involved in the regulation of a variety of eukaryotic genes and is formed in higher eukaryotes by three subunits NF-YA/B/C. We have characterized NF-Y of the trematode parasite Schistosoma mansoni and studied the structure and the function of the SMNF-YA subunit. In this work, we present the cloning and sequence analysis of the B subunit of the parasite factor. SMNF-YB contains the conserved HAP-3 homology domain but the remaining part of the protein was found to be highly divergent from all other species. We demonstrated by transfections of GAL4 fusion constructs, that mouse NF-YB does not contain activation domains while the C-terminal part of SMNF-YB has transcriptional activation potential. On the other hand, the N-terminal parts of SMNF-YA and mouse NF-YA were shown to mediate transactivation; the integrity of a large 160 amino acid glutamine-rich domain of NF-YA was required for this function and an adjacent serine- and threonine-rich domain was necessary for full activity in HepG2, but redundant in other cell types. Transactivation domains identified in SMNF-YB are also rich in serine and threonine residues. Our results indicate that serine/threonine-richsequences from helminth parasites potentiate trans-cription and that such structures have diverged during evolution within the same transcription factor.