Anatomy of Highly Expressing Chromosomal Sites Targeted by Retroviral Vectors

Abstract

The eukaryotic genome contains chromosomal loci with a high transcription-promoting potential. For their identification in cultured cells, transfer of a reporter gene has to be performed by a technique that grants the integration of individual copies. We have applied retroviral vectors in conjunction with inverse polymerase chain reaction techniques to reconstruct a number of these sites for a further characterization. Remarkably, all examples conform to the same design in that the process of retroviral infection selected a scaffold- or matrix-attached region (S/MAR) that was flanked by DNA with high bending potential. The S/MARs are of an unusual type in that they show a high incidence of certain dinucleotide repeats and the potential to act as topological sinks. The anatomy of retroviral integration sites reveals principles that can be exploited for the development of predictable transgenic systems on the basis of expression and targeting vectors.

The Brf and TATA-binding Protein Subunits of the RNA Polymerase III Transcription Factor IIIB Mediate Position-specific Integration of the Gypsy-like Element, Ty3

“…Ty3 introduces its strand transfers at nucleotides Ϫ5 and Ϫ1 (thus inserting Ty3 sequence beginning at bp Ϫ6), adjacent to the downstream end of DNA-bound Brf (42), and within the domain of the transcription bubble (39). Retroelements preferentially mediate integration into regions of DNA containing bends or kinks (47)(48)(49)(50). The work that is presented here suggests that nucleophilic attack mediated by Ty3 integrase resembles the process mediated by retroviral integrase proteins in the sense that it favors positions with distorted base pairing or stacking but that, in the case of Ty3, protein-protein interactions play a dominant role, perhaps by converting a basal activity for nonspecific insertion to locationspecific function.…”

Bcl-X is the major pleiotropic anti-apoptotic gene activated by retroviral insertion mutagenesis in an IL-3 dependent bone marrow derived cell line

“…In vivo, in the absence of selection, it seems that all regions of the genome are accessible to retroviral integration, although some sites are used at a higher frequency than expected for random integration (Engelman, 1994). It is believed that the DNA structure but not the sequence of these integration hot spots could favour retroviral insertion (Engelman, 1994;Muller and Varmus, 1994;Mielke et al, 1996). An integration preference for actively transcribed DNA or DNAse I hypersensitive sites has also been described (Engelman, 1994).…”

Scaffold/Matrix Attachment Regions (S/MARs): Relevance for Disease and Therapy

“…This activity appears to occur at a distinct subset of sites, as externally added S/MAR sequences and ssDNA do not interfere with the process. In contrast, there is a competition between ssDNA binding and prototype S/MAR binding on some scaffold-associated proteins, but not on others (Kay and Bode 1994;Mielke et al 1996). In retrospect, competition patterns have proven valuable as they can be applied to reveal specific binding modes.…”