Identification and analysis of a matrix‐attachment region 5′ of the rat glutamate‐dehydrogenase‐encoding gene

Abstract

Eukaryotic chromatin is thought to be organized into independently regulated loop domains by interaction of matrix‐attachment regions (MAR) of the DNA to the nuclear matrix. To define the borders of the chromatin loop containing the glutamate dehydrogenase (GDH) gene, we screened the GDH gene and flanking regions for the presence of MAR sequences. We here report identification, mapping and sequencing of an (A+T)‐rich MAR located 2010–1397 bp upstream of the transcription initiation site of GDH, that mediates strong binding to the nuclear matrix. Smaller regions can also confer binding capacity, although at a lower affinity. This (A+T)‐rich MAR contained 11 bp and 12 bp (A+T)‐rich direct repeats, but not any of the sequences previously described to be associated with MAR activity. We here show that the presence of (A+T)‐rich domains of DNA is not sufficient to confer binding capacity, since (A+T)‐rich sequences located downstream of the identified MAR did not bind to the nuclear matrix. Moreover, a consensus topoisomerase‐II‐binding site located downstream of the MAR was found to be insufficient to mediate substantial binding. The number of binding sites in the nuclear matrix for MAR‐containing fragments was shown to be approximately 15 000/nucleus. Since organization of the entire rat genome in loops with an average loop size of 100 kbp would require 60 000 binding sites, this suggests that only part of the genome is organized in loops. Alternatively, we might have underestimated the number of binding sites. The GDH MAR, and MAR‐containing fragments derived from other species, were found to bind to the same binding sites in the nuclear matrix, although the affinity varied.

NuMA after 30 years: the matrix revisited

“…Following from these early ideas, it has been proposed that the organization of DNA in the nucleus has cell and tissue specific determinants and that differential organization contributes to tissue specific gene expression [75]. Consistent with a role in genomic organization, tightly regulated NuMA levels have been proposed to be essential for the creation of chromosome domains either by organizing chromatin [76], by interacting with specific DNA domains called matrix attachment regions (MARs) [77, 8] or by simply providing a substrate for intranuclear processes (Table 1). Yeast two-hybrid studies suggested an interaction between NuMA and GAS41 ( g lioma- a mplified- s equence 41) [78], an ubiquitous, essential transcription factor amplified in human gliomas [9].…”

Genomewide identification of nuclear matrix attachment regions: an analysis of methods

“…These two characteristics alone are not sufficient for nuclear matrix binding [32,33]. Just as the classification of the general characteristics of MARs has remained elusive, so has the identification of a consensus sequence.…”

Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove.

“…Figure 6 shows the results of our investigation of binding to lamin of different MAR and non-MAR sequences. We studied competition of the Drosophila histone MAR with the MAR of the mouse immunoglobulin K light chain locus (10,11), the 5' MAR of the chicken lysozyme gene (38), the 5' MAR of the rat glutamate dehydrogenase gene (14), and the synthetic polynucleotides poly(dA-dT) and poly(dA) -poly(dT). We found that random non-MAR DNA, i.e., pBR322 fragments, competed only poorly.…”