Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta

Abstract

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.

Mechanisms underlying lack of insulin-like growth factor-binding protein-3 expression in non-small-cell lung cancer

“…To investigate the effects of IGFBP-3 promoter methylation on the binding of Sp-1 and other transcription regulators such as MeCP2, and HDAC in NSCLC cells (Zhang et al, 1986;Nan et al, 1997Nan et al, , 1998Jones et al, 1998), we performed ChiP assays in a high-IGFBP-3-expressing NSCLC cell line with an unmethylated promoter (H460 cells) and an IGFBP-3-negative NSCLC cell line with a methylated promoter (H1299). In H460 cells, strong binding of the Sp-1, but not of the MeCP2 and HDAC, to IGFBP-3 promoter was observed, which were not affected by 5 0 -aza-dC treatment ( Figure 6).…”

“…The discovery of methylation-dependent DNA-binding proteins such as methyl-CpG-binding protein-2 (MeCP2) suggests that transcriptional repression by methylation may be due in part to the binding of transcription factors or the action of the proteins themselves as transcription repressors (Zhang et al, 1986;Nan et al, 1997). Nan et al 1997 were able to identify a region capable of transcription repression in vivo -the transcription repression domain (TRD).…”

A murine ATFa-associated factor with transcriptional repressing activity

“…Proteins have been characterized that modulate the chromatin state and may thereby negatively act on template accessibility to the transcription machinery. These include the silencing proteins in yeast (Orlando and Paro, 1995), the Polycomb group gene products in Drosophila (Simon, 1995) or the methylated-DNA-binding proteins in vertebrates (Zhang et al, 1986;Nan et al, 1997) all of which create a repressive chromatin state, as opposed to multiprotein complexes like the yeast SWI/SNF and their metazoan homologues (Winston and Carlson, 1992;Tsukiyama and Wu, 1995) which increase nucleosome mobility (Kingston et al, 1996;Kadonaga, 1998).…”

Methylation adjacent to negatively regulating AP-1 site reactivates TrkA gene expression during cancer progression

“…Previously, only a limited range of transcription factors such as E2F (Campanero et al, 2000) was considered to be affected by methylation because the CpG dinucleotide sequence within the binding site was thought to be essential. More recently, however, we (Kitazawa et al, 1999) and others (Zhu et al, 2003) have reported that CpG methylation adjacent to transcription factor-binding sites can either directly or indirectly through methylation-dependent, sequence-specific DNA-binding proteins (Zhang et al, 1986) also affect the DNA binding of transcription factors. Therefore, CpG methylation in non-CpG islands, the role of which has not yet been well evaluated in cancer progression, could contribute to minimum alteration of the specific gene expression at the transcription level by altering the affinity of the transcription factors to the DNA.…”