Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

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 two- to 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.

Nuclear matrix protein ARBP recognizes a novel DNA sequence motif with high affinity.

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.

Biochemical properties of attachment region binding protein ARBP.

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 filtration 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.

Stimulation of the biosynthesis of lactosamine repeats in glycoproteins in differentiating U937 cells and its suppression in the presence of NH4Cl.

We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.