Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms

Abstract

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island

“…This process commonly called "gene silencing" has been found to be dysregulated following cellular activation. For many genes, hypermethylation of CpG islands is associated with delayed replication, condensed chromatin, and gene silencing (4,5,20,43). Transcriptional repressor proteins that have been shown to be functional in this process include the methyl-CpG-binding proteins-1 and -2 (MeCP1, MeCP2) and methyl-CpG binding domain family members (MBD1, MBD2a, MBD2b, and MBD4) (6,22,44).…”

Methyl CpG binding proteins: coupling chromatin architecture to gene regulation

“…Recombinant MBD1 can repress transcription of methylated, but not unmethylated, templates in vitro much like MeCP2 (Cross et al, 1997;Fujita et al, 1999). The various isoforms of MBD1 repress transcription in both mammalian and insect cells Ng et al, 2000).…”

Methyl-CpG Binding Domain Proteins and Their Involvement in the Regulation of the MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 Gene Promoters

“…MBD1 is alternatively spliced to produce five protein isoforms (PCM1, MBD1v1, MBD1v2, MBD1v3, and MBD1v4) which differ in the number of cysteine-rich (CXXC) domains and the carboxyl-terminal sequence. All five MBD1 isoforms repress transcription from methylated promoters and the variants with three CXXC domains additionally repress unmethylated promoters (26,27). Two isoforms of MBD2 are known: MBD2a and MBD2b.…”