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, S urprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spann ing 125 amino acids in the N-terminal halves is 96.8% identical betwee n chicken ARBP and rat MeCP2. A deletion mutation analysis using South western and band shift assays identified this highly conserved region as the MAR DNA binding domain, Alignment of chicken ARBP with rat: and human McCP2 proteins revealed six trinucleotide amplifications genera ting up to 34-fold repetitions of a single amino acid, Because MeCP2 w as previously localized au pericentromeric heterochromatin in mouse ch romosomes, we analyzed the in vitro binding of ARBP to various repetit ive sequences, In band shift experiments, ARBP binds to two chicken re petitive sequences as well as to mouse satellite DNA with high affinit y similar to that of its binding to chicken lysozyme MAR fragments, In mouse satellite DNA, use of several footprinting techniques character ized two high-affinity binding sites, whose sequences are related to t he ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3 '). Band shift experiments indicated that methylation increased in vit ro binding of ARBP to mouse satellite DNA two- to fivefold, Our result s suggest that ARBP/McCP2 is a multifunctional protein with roles In l oop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.