In vitro detection of methylated DNA via recombinant protein MBD2b

Abstract

Members of the methyl binding domain (MBD) protein family are known for binding to methylated DNA by recognizing methylated cytosines. Their original function is to regulate protein biosynthesis by recruitment of transcriptional repression complexes to silence gene expression. The aim of the presented work was to detect methylated DNA spotted onto nitrocellulose membranes with recombinant proteins MBD2b, MBD2b-GFP and directly labeled protein MBD2b. Proteins were affinity purified and tested for functionality before application. We were able to show that these functional recombinant proteins bind to unilaterally and symmetrically methylated oligonucleotides and genomic DNA in vitro and thus can be used in various detection assays.

Epigenetic silencing of serine protease HTRA1 drives polyploidy

“…3c ). These results are in agreement with published data reporting that MBD2 binds to synthetic 34mer nucleotides with high affinity [ 21 ]. To obtain independent evidence for binding of MBD2 to HTRA1 promoter DNA, we performed proteolytic digests of MBD2 without or with bound DNA (Fig.…”

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

“…The methylated DNA signal is read out by the methylCpG-binding domain (MBD) proteins [5,6]. These proteins can recognize and bind methylated CpG sites to mediate transcriptional repression via recruitment of co-repressors such as histone deacetylases (HDAC), leading to chromatin condensation [7,8].…”

“…Mutations in the MBD proteins can contribute to human diseases, such as Rett syndrome, a severe X-linked mental retardation that afflicts about one in 10000 girls [2]. Among all the MBD family members (MBD1, MBD2, MBD3, MBD4 and MeCP2), MBD2b protein, a small fraction of the total MBD2 proteins lacking 140 N-terminal aa [5,6], has the highest affinity for methylated DNA and displays the greatest capacity to differentiate between methylated and unmethylated DNA [9]. Based on the binding property of MBD, a series of analytical methods for detection of DNA methylation were established, such as methylated-CpG island recovery assay (MIRA) [10], MBD-isolated genome sequencing (MiGS) [11], and MBD column chromatography [12][13][14][15].…”

Interplay of Binding Stoichiometry and Recognition Specificity for the Interaction of MBD2b Protein and Methylated DNA Revealed by Affinity Capillary Electrophoresis Coupled with Laser-Induced Fluorescence Analysis

“…12,22 Recognition of the methyl-CpG pair depends on the hydrophobic interactions between the methyl groups of 5-methylcytosines and a hydrophobic patch formed by five residues, Val-20, Arg-22, Tyl-34, Arg-44, and Ser-45. 9,12,23 Subtle variations in this hydrophobic network might abolish the binding of the protein to methylated DNA. For example, the loss of a single hydroxyl group Y34F renders MBD3 incapable of binding to methylated DNA.…”

“…One of the most important epigenetic modifications in mammalian cells is DNA methylation. − It occurs predominantly on CpG dinucleotides. Human gen promoters of about 60% are associated with CpG islands, which display high CpG density and are usually free of methylation in healthy cells. − , Dynamic DNA methylation/demethylation regulates many cellular processes, including nuclear reprogramming, transcription, genomic imprinting, and X-chromosome inactivation. − Dysregulation of DNA methylation occurs in many cancer cells, in which the hypermethylation of promoter CpG islands may inactivate some tumor suppressor genes. , Methyl-CpG binding domain proteins (MBD) are considered central players in DNA methylation-dependent gene silencing. − ,− These proteins can bind methylated CpG and further recruit corepressors, such as histone deacetylases (HDAC), to establish silent chromatin, thus providing a link between DNA methylation and transcriptional repression. ,− …”