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.

Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.

To study the influence of clustered highly repetitive DNA sequences on the expression of adjacent genes, LTK- cells were cotransfected with the herpes simplex virus thymidine kinase (tk) gene and mouse satellite DNA. TK+ transformants containing a few copies of the tk genes flanked by satellite DNA were isolated. In situ hybridization on the metaphase chromosomes indicated that in each cell line the TK sequences resided at a single chromosomal site and that integration occurred preferentially into regions of the cellular DNA rich in highly repetitive sequences. The prominent feature of these cell lines was their phenotypic instability. Suppression and reexpression of the tk gene occurred at high frequency (greater than 3%) and did not correlate with any significant change in the organization of foreign DNA or with the presence of selective agents. These results indicate that satellite DNA, the major component of constitutive heterochromatin, may influence the expression of adjacent genes by affecting the chromatin structure.