We have transfected DNA corresponding to the complete chromatin domain of human interferon beta (huIFN-beta) gene into mouse L cells. In this construct, which is flanked by scaffold-attached regions (SARs), the gene's transcription was enhanced 20-30-fold with respect to DNAs containing only the immediate regulatory elements. To elucidate the role of SAR elements in the transcriptional enhancement, their position was varied relative to several artificial promoter-gene combinations. It was found that SARs enhance general promoter functions in an orientation- and partially distance-independent manner; their effect is restricted to the integrated state of transfected templates. During the phase of transient expression, SAR elements were generally found to have an antagonizing effect.

Regions attached to the nuclear scaffold have been traced after transfecting a 36-kilobase (kb) piece of DNA, surrounding the human interferon-beta gene, into mouse L-cells. An extended attached region starts 1.7 kb upstream from the gene and a moderate binding site immediately downstream. These findings could be confirmed by reconstitution experiments in vitro which predict another scaffold-attached region (SAR) starting 12 kb downstream from the gene. Since no other transcripts originate from DNA between the major SARs, these elements could be involved in interferon gene regulation.

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.

Scaffold/matrix-attached regions (S/MARs) are cis-acting elements with a function outside transcribed regions and in introns. Although they usually augment transcriptional rates, their action is highly context-dependent. We cloned an 800 bp S/MAR element from the upstream border of the human interferon-beta domain at various positions within a transcribed region of 4.3 kb. By use of retroviral gene transfer, the vector could be integrated into target cells as a single copy enabling a rigorous definition of the distance between the S/MAR and the transcriptional start site. At a distance of about 4 kb, the S/MAR supported transcriptional initiation, whereas at distances below 2.5 kb, transcription was essentially shut off. Controls proved the functionally of all constructs in the transient expression phase and ruled out any influence of S/MAR position on transcript stability. Moreover, no pausing or premature termination was observed within these elements. We suggest that the protein binding partners of S/MARs change according to the topological status, explaining these divergent S/MAR effects.

SATB1 specifically recognizes and binds to specialized genomic regions with an ATC sequence context with high base-unpairing propensity. Such base-unpairing regions (BURs) are typically identified within nuclear scaffold- or matrix-attachment regions (S/MARs). SATB1 is a homeodomain protein and is predominantly expressed in thymocytes. We obtained BHK cell lines expressing low levels of SATB1 by stable transfection and investigated its effect on stably integrated MAR-linked SV40 enhancer/promoter-driven luciferase reporter genes. For this study, both naturally occurring and synthetic MARs, as well as an AT-rich non-MAR control, were tested. Previous studies demonstrated that MAR sequences augment transcription of the linked reporter luciferase gene. Here, we show that SATB1 dramatically reduces the high levels of MAR-linked luciferase gene transcription. Transcription was virtually abolished for a reporter gene surrounded by two MARs at the 5' and 3' ends of the gene, which otherwise confer the highest level of transcriptional augmentation. On the other hand, SATB1 did not affect expression of an AT-rich non-MAR-linked luciferase gene or of endogenous housekeeping genes. This study shows that SATB1 acts as a strong transcriptional suppressor on a reporter gene linked to MARs when it is stably integrated into chromatin.