MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

Abstract

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.Addition of methyl groups mostly at the CpG dinucleotide represents the major epigenetic modification of eukaryotic genomes heritable by somatic cells after cell division. DNA methylation plays an essential role in mammalian development (24) and in different biological processes such as tissue-specific gene expression (40), X-chromosome inactivation (17), genomic imprinting (2, 33), and repression of transposable elements (46). Abnormal methylation is a cause of human genetic diseases, including ICF syndrome (16), and is involved in carcinogenic processes primarily through aberrant hypermethylation of tumor suppressors' promoter regions (12,21,22).Methylated DNA is generally associated with transcriptional silencing (6, 7, 32), but how DNA methylation silences gene expression is not well understood. Studies are now focusing on the different components of the DNA methylation system and particularly on the mechanisms by which methyl-CpG signal is targeted, read, and maintained. Recently, a family of five mammalian methyl-CpG-binding proteins (MeCP2, MBD1, MBD2, MBD3, and MBD4) has been found to be essential to interpret the methylation patterns and to mediate the biological consequences of DNA methylation (1, 15, 18). All methyl-binding proteins share a common structural stretch of 60 to 80 amino acids called the mCpG-binding domain (MBD) and, with the exception of MBD4 (19, 34), mediate transcription...

POZ-, AT-hook-, and Zinc Finger-containing Protein (PATZ) Interacts with Human Oncogene B Cell Lymphoma 6 (BCL6) and Is Required for Its Negative Autoregulation

“…Human heart and placenta cDNA libraries (Clontech) were simultaneously analyzed. A total of about 2 ϫ 10 6 clones were tested for each library, and the specificity of interaction was assessed as described previously (16).…”

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

“…The shorter form, MBD2b, starting at the second methionine therefore lacks the NH 2 -terminal sequence of MBD2a (28). Recent reports described MBD2a as both an activator and a repressor of transcription (29)(30)(31). Mbd1-and Mbd2-deficient mice are viable and fertile.…”

Other Proteins Interacting with XP Proteins

“…32 Other XPA interacting proteins include ATR (ATM and RAD3-Related), 33 a DNA damage checkpoint kinase of the phosphoinositide 3-kinase-like kinase (PIKK) family, XAB1 (XPA-binding protein 1) 34,35 and XAB2 (XPA-binding protein 2). 36,37 The interaction of XPA with ATR may be responsible for the rapid translocation of XPA from the cytoplasm to the nucleus in response to UV irradiation as this activity is dependent on ATR and can be abolished using either ATR inhibitors or siRNA-mediated knockdown of the kinase. 33 Although the mechanism of the translocation remains unclear, it is possible that the XPA binding protein XAB1, a cytoplasmic GTPase, may be involved.…”

“…34 XAB2 interacts with a variety of proteins including RNA Pol II and is active in mRNA splicing. 36,37 While its role in NER is not quite clear, it is believed that it promotes TC-NER through its dual interaction between RNA Pol II and XPA. However, attempts to study this protein interaction in vivo using transgenic mice have proven difficult as deletion of XAB2 is embryonically lethal.…”

“…However, attempts to study this protein interaction in vivo using transgenic mice have proven difficult as deletion of XAB2 is embryonically lethal. 36 …”