A locus located downstream of N-myc2 and targeted by WHV integration in woodchuck liver tumors harbors S/MAR elements

R Bruni MSc^a, E D'Ugo MSc^a, U Villano MSc^a, G Fourel PhD^b, MSc La Sorsa^a, MSc Conti^a, M-A Buendia PhD^b, M Rapicetta PhD^a

^aLaboratory of Virology, Istituto Superiore di Sanità, Rome, Italy, ^bUnités d’ Oncogenèse et Virologie Moléculaire, Département de Virologie, INSERM U579, Institut Pasteur, Paris, France

AIM. Woodchuck Hepatitis Virus (WHV) and the woodchuck (Marmota monax) is a model for hepatocellular carcinoma induced by Hepatitis B Virus (HBV). In woodchuck liver tumors the N-myc2 proto-oncogene is frequently activated by WHV integration either close to the gene or in the b3n and win downstream loci, located 10 kb and 150 kb from N-myc2, respectively. A Scaffold/Matrix Attachment Region (S/MAR) was shown to be located in b3n near WHV insertion sites. As S/MARs are involved both in organization of chromosomal DNA in functional domains and in the regulation of gene expression, the b3n S/MAR was hypothesized to mediate activation of the upstream N-myc2 gene upon WHV integration. Aim of the present work was to investigate if S/MAR elements are located in win too. METHODS. The woodchuck win region was cloned by phage library and sequenced by a M13 shotgun method. Candidate S/MAR regions were predicted by search for S/MAR motifs in the win sequence by programs of the GCG package. Overlapping fragments spanning candidate regions as well as a region poor of S/MAR motifs were amplified by PCR from M13 sub-clones. The affinity of these fragments for nuclear scaffold was tested in a standard in vitro binding assay commonly used to investigate if a DNA fragment is a S/MAR. RESULTS. Sequence analysis of a cloned 17 kb DNA fragment corresponding to the major region of WHV insertion in win showed three regions with high density of S/MAR motifs. High affinity for nuclear scaffold was shown by fragments from one of them, as well as by fragments from a region poor of S/MAR motifs. On the whole, results suggest that at least two S/MAR elements are present in win: a small one (0.3 kb), a member of a minor class of S/MARs, poor of S/MAR peculiar motifs, and a larger one (1.75 kb), a typical member of the largely represented AT-rich class of S/MAR elements. CONCLUSIONS. Our present and previous findings show that both loci downstream of N-myc2 targeted by WHV integration harbor S/MARs. These findings suggest that these control elements might be involved in the mechanism of N-myc2 activation triggered by WHV insertion in these loci. The presence of S/MARs at the integration sites of several oncogenic viruses, including human HBV and HPV and mouse Murine Leukemia Virus (MuLV), has also recently been reported, suggesting a general role for viral integration close to S/MARs in tumor development. Despite differences between woodchuck and human HCC as for cellular genes affected by WHV and HBV integration, respectively, our findings also suggest that some molecular mechanisms underlying cellular gene activation may be shared by WHV and HBV integrations.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 791 (Viral Infections).