No abstract

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5-GGTGT-3). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA twoto fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.

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.

ARBP (attachment region binding protein) is an abundant nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/ SARs). Here we show by gel tiltration and gradient sedimentation that ARBP has an elongated shape. The sedimentation coefficient was determined as only 2.1 S. Furthermore, limited proteolysis of ARBP in situ (in isolated nuclei) with several proteases generated limiting resistant peptides from 14.5 to 18 kDa, that retained the ability to bind MARS specifically. This indicates that these peptides encompass the DNA binding domain of ARBP.