Computational and in Vitro Analysis of Destabilized DNA Regions in the Interferon Gene Cluster:  Potential of Predicting Functional Gene Domains

Abstract

Recent evidence adds support to a traditional concept according to which the eukaryotic nucleus is organized into functional domains by scaffold or matrix attachment regions (S/MARs). These regions have previously been predicted to have a high potential for stress-induced duplex destabilization (SIDD). Here we report the parallel results of binding (reassociation) and computational SIDD analyses for regions within the human interferon gene cluster on the short arm of chromosome 9 (9p22). To verify and further refine the biomathematical methods, we focus on a 10 kb region in the cluster with the pseudogene IFNWP18 and the interferon R genes IFNA10 and IFNA7. In a series of S/MAR binding assays, we investigate the promoter and termination regions and additional attachment sequences that were detected in the SIDD profile. The promoters of the IFNA10 and the IFNA7 genes have a moderate ∼20% binding affinity to the nuclear matrix; the termination sequences show stronger association (70-80%) under our standardized conditions. No comparable destabilized elements were detected flanking the IFNWP18 pseudogene, suggesting that selective pressure acts on the physicochemical properties detected here. In extended, noncoding regions a striking periodicity is found of rather restricted SIDD minima with scaffold binding potential. By various criteria, the underlying sequences represent a new class of S/MARs, thought to be involved in a higher level organization of the genome. Together, these data emphasize the relevance of SIDD calculations as a valid approach for the localization of structural, regulatory, and coding regions in the eukaryotic genome.

Performance of Genomic Bordering Elements at Predefined Genomic Loci

“…25,2005 GENOMIC BORDERING 2263 on May 9, 2018 by guest http://mcb.asm.org/ (IS) have been identified by a biomathematical prediction approach (SIDD) and were characterized by competition with prototype S/MARs (24). ISs mediate a strong attachment to both murine and human scaffolds; as prototype S/MARs, they show a strong augmentation effect after stable integration.…”

“…A total of 20 l of the extracts was added to 350 l of reaction buffer (25 Quantification of S/MAR activities in vitro (halo reassociation). In vitrobinding assays of DNA sequences to lithium salt (LIS)-extracted nuclear matrices were carried out and quantified according to the modified equal counts approach as previously described (24,31). This procedure includes a stringent control of the scaffold-binding potential.…”

“…Alternatively, a statistical mechanical procedure was applied to derive SIDD profiles that identify regions of DNA unwinding and, thereby, secondary-structure-forming potentials (7). There are many cases where the prediction of these two approaches is in close agreement (24). FIG.…”

“…The latter procedure was pioneered by Cockerill and Garrard (14). To date, several variants of this protocol are available, permitting the precise quantification of the interaction strength (24,31), which is a parameter that lends itself to computer-assisted predictions (7). Subsequently, domain-bordering activities as well as multiple S/MAR-enhancer interactions (both negative and supportive) have been reported; the latter class of activities is outside the scope of this contribution.…”

Use of human MAR elements to improve retroviral vector production

“…26 For instance, the scaffold/matrix attachment regions (MARs) are thought to associate to the nuclear matrix, 27,28 and to organize eukaryotic chromatin into independent loops. [29][30][31][32] The establishment of independently regulated domains by loop formation has been proposed to insulate transgenes from negative effects of the genomic surroundings. 26,29 Alternatively, MARs may boost the function of transcriptional enhancers, 29,[33][34][35] and they may act as origins of replication when transcribed.…”