1GBF - German Research Center for Biotechnology / Epigenetic Regulation,
D-38124 Braunschweig
2The Mount Sinai Med. Center, New York,
NY 10029, USA;
3Wayne State University School of Medicine,
Detroit, MI 42801, USA;
The human interferon gene cluster on the short arm of chromosome 9 comprises 26 genes the functional members of which are separated by highly efficient scaffold/matrix attached regions (S/MARs). S/MARs have been discovered more than a decade ago and have been defined as DNA-elements staying attached to or associating with the nuclear skeleton after the extraction of the histones and soluble factors from eukaryotic nuclei. While S/MARs do not conform to any obvious sequence consensus, their recognition is governed by structural features, most significantly a propensity to expose single strands under negative superhelical tension. To predict the activity of these elements we have refined various in vitro and biomathematical methods including LIS-extraction and stress-induced duplex-destabilization (SIDD) profiles. Other features of individual chromatin domains which become apparent in SIDD profiling are the localization of regulatory elements (promoters, terminator sequences and DNAse I hypersensitive sites) and of chromosomal breakpoints which explain the genomic instability of this genomic locus.
To verify and further to improve methods of biomathematical profiling we focused on a 10 kb region in the human interferon gencluster including the pseudogene IFNWP18 and the interferon alpha genes IFNA10 and IFNA7. In a series of S/MAR-binding assays with LIS-extracted nuclei we investigated the promotor and terminator regions of these genes and additional destabilized sequences detected by SIDD analysis. These binding experiments revealed no extended SAR-elements in the analyzed domain. The promotors of as well the IFNA10 and the IFNA7 genes have an apparent affinity to the nuclear matrix of about 20%, the terminator sequences of both of these genes show association values of 40-60%. Suprisingly, no such destabilised elements could be detected flanking the IFNWP18 pseudogene. Basically, the in vitro data confirmed what was predicted by biomathematical calculations. In conclusion eukaryotic promotor- and terminator regions have a destabilized character but are not what has usually been considered to be a S/MAR.