Nuclear Matrix Protein ARBP Recognizes a Novel DNA Sequence Motif with High Affinity

Abstract

ARBP is a nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/SARs). Here we characterize by DNase I footprinting, dimethyl sulfate protection, and mobility shift assays two binding sites for ARBP within a chicken lysozyme MAR fragment. Our results indicate that ARBP recognizes a novel DNA sequence motif containing the central sequence 5'-GGTGT-3' and flanking AT-rich sequences. Binding occurs through major groove contacts to two guanines of the central sequence. Collective and single-base substitutions in the 5'-GGTGT-3' core motif result in loss or significant reductions of ARBP binding, underscoring the importance of the GC-rich core sequence. Structural elements of the sequence motif are probably also recognized. The affinity of ARBP to both binding sites is surprisingly high [KD = (2-6) x 10(-10) M]. High-affinity recognition of the identified DNA motif in MARs/SARs by ARBP is likely an important feature in the domain organization of chromatin.

Chicken MAR-Binding Protein ARBP Is Homologous to Rat Methyl-CpG-Binding Protein MeCP2

“…Studies of a number of prokaryotic and eukaryotic DNA-binding proteins have demonstrated that insertion of an ␣-helix into the major groove is a frequent mode of binding. It was previously shown that ARBP recognizes a specific motif in the lysozyme MAR through contacts in the major groove (5). Methylation protection experiments furthermore indicate that recognition of specific base pairs in the major groove is significant for binding of ARBP to two high-affinity binding sites in mouse satellite DNA as well (see below and Fig.…”

“…Summarizing our footprint data, we first point out that the results did not reveal the seven CpGs of the sequence as high-affinity binding sites but revealed two sites, I and II, that have similarities to the ARBP binding site core consensus in the lysozyme 5Ј MAR (5). Only one of these sites (site I) contains a CpG in its center.…”

“…While some MAR-binding proteins seem to have additional roles in RNA processing and transport, others function mainly as structuring proteins at the level of higher-order chromatin organization. The abundant nuclear protein ARBP (attachment region binding protein), a member of the latter subgroup, specifically binds to MAR/SARs in the presence of excess unspecific competitor DNA and recognizes in the lysozyme 5Ј MAR a defined sequence motif through contacts in the major groove as well as the minor groove (5,47). This motif contains the central sequence 5Ј-GGTGT-3Ј and AT-rich flanking sequences.…”

“…Southwestern assays employing the lysozyme 5Ј MAR fragment H1-HaeII as a probe in the presence of 50 g of E. coli competitor DNA per ml were performed as described previously (47). Electrophoretic mobility shift assays, DNase I footprint analyses, and dimethylsulfate (DMS) protection assays using purified ARBP (gift of J. P. von Kries) were performed as described elsewhere (5). The percentage of bound DNA in band shift assays was determined by scanning the autoradiogram.…”

Role of Nuclear Matrix in Estrogen Regulated Gene Expression in Human Breast Cancer Cells

“…MAR-binding proteins include lamins, which are found in the nuclear pore-lamina and internal matrix , topoisomerase II, SATB1, HET (SAF-B), and attachment region binding protein which is an internal matrix protein or nuclear matrin (Pommier et al, 1990;Nakayasu and Berezney, 1991;von Kries et al, 1991;Luderus et al, 1992;Buhrmester et al, 1995;.…”