Engineering a high-affinity methyl-CpG-binding protein

Jorgensen, H. F.

doi:10.1093/nar/gkl527

Cited by 78 publications

(76 citation statements)

References 27 publications

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“…Because MBD2 has a methyl-CpG binding domain, we tested to what extent MBD2 binding to DNA is dependent on methylation. To this end, we performed a methylated DNA precipitation on chip (MAP-chip), using a recombinant protein (4xMBD) consisting of four methyl-CpG binding domains derived from MBD1 31 to selectively precipitate methylated sequences. MAP is a highly specific and sensitive technique for selecting methylated other factors, and may thus play a dual role.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmids pET-4xMBD and pET-4xMBD-R22A, encoding respectively the recombinant wild-type His 6 -tagged 4xMBD and mutant His 6 -tagged 4xMBD-R22A in a bacterial system, were kindly provided by Dr. A. Bird. 31 Both recombinant proteins were purified from induced E. coli Rosetta (DE3 pLysS) cultures on Ni-NTA agarose (Qiagen, Courtaboeuf, were quantitated by Q-PCR. These were the BRCA1, 14 hTERT, 15 pS2, 29 PARVG, 18 RASSF1A 18 and GAPDH promoters.…”

Section: Methodsmentioning

confidence: 99%

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Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions

Chatagnon

Perriaud²,

Nazaret³

et al. 2011

Epigenetics

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions

Chatagnon

Perriaud²,

Nazaret³

et al. 2011

Epigenetics

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“…Because AtMBD7 is unique in having three MBD motifs, we tested the significance of this feature for localization and binding affinity to its chromosomal sites. Recently, it has been shown that a recombinant poly-MBD protein made of the MBD motif of the mammalian Mbd1 has higher binding affinity to mCpG sites than the monomeric Mbd1 protein (10). This finding suggested that the naturally occurring poly-MBD protein AtMBD7 might display higher binding affinity for methylated sites than the monomeric AtMBD5 and AtMBD6 proteins.…”

Section: Resultsmentioning

confidence: 99%

The Three Methyl-CpG-binding Domains of AtMBD7 Control Its Subnuclear Localization and Mobility

Zemach

Gaspan

Grafi

2008

Journal of Biological Chemistry

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Three methyl-CpG-binding domain (MBD) proteins in Arabidopsis, AtMBD5, AtMBD6, and AtMBD7, are functional in binding methylated CpG dinucleotides in vitro and localize to the highly CpG-methylated chromocenters in vivo. These proteins differ, however, in their subnuclear localization pattern; AtMBD5 and AtMBD6, each containing a single MBD motif, show preference for two perinucleolar chromocenters, whereas AtMBD7, a naturally occurring poly-MBD protein containing three MBD motifs, localizes to all chromocenters. Here we studied the significance of multiple MBD motifs for subnuclear localization and mobility in living cells. We found that the number of MBD motifs determines the subnuclear localization of the MBD protein. Furthermore, live kinetic experiments showed that AtMBD7-green fluorescent protein (GFP) has lower mobility than AtMBD5-GFP and AtMBD6-GFP, which is conferred by cooperative activity of its three MBD motifs. Thus, the number of MBD motifs appears to affect not only binding affinity and mobility within the nucleus, but also the subnuclear localization of the protein. Our results suggest that poly-MBD proteins can directly affect chromatin structure by inducing intra-and interchromatin compaction via bridging over multiple methylated CpG sites.Cytosine methylation is a common epigenetic modification of most eukaryotic nuclear DNA. Intensive studies demonstrated the central role played by cytosine methylation in genome organization, gene expression, and growth and development. The way by which the methyl group is interpreted into a functional state has begun to be unraveled with the isolation and characterization of methylated DNA-binding proteins. This group of proteins includes an evolutionary conserved protein family, initially described in animals, termed methyl-CpGbinding domain (MBD) 2 proteins (1). Biochemical and molecular analyses suggest that mammalian MBDs induce the formation of a repressive chromatin structure at methylated CpG (mCpG) sites by the recruitment of various chromatin factors including histone deacetylases, histone methyltransferases, and ATPase-dependent nucleosome remodeling factors (2, 3). Thus far, of the 13 AtMBD proteins found in Arabidopsis, only AtMBD5, AtMBD6, and AtMBD7 were found to bind mCpG sites in vitro and to localize in vivo to the highly methylated chromocenters (ChCs) (reviewed in Refs. 4 and 5). This chromocenter localization is CpG methylation-dependent inasmuch as it was disrupted in the hypomethylated mutants ddm1 and met1 (6). Yet, these three AtMBDs display different localization patterns within Arabidopsis nuclei; AtMBD7 is predominantly localized at all ChCs, whereas AtMBD5 and AtMBD6 show preference for two perinucleolar ChCs adjacent to the ribosomal DNA clusters (6). A major structural feature distinguishing AtMBD7 from AtMBD5 and AtMBD6 is the presence of three MBD motifs within its protein sequence. Notably, naturally occurring poly-MBD proteins appear to be unique to plants as to date no such protein has been found in animals. Here we studied...

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“…These molecules were selected from a mixture that included unmethylated DNA and were recognized using a fluorescently labeled methyl binding domain protein-1 (MBD1) that binds specifically to double-stranded, methylated DNA (24). We discuss the technologies that enabled us to identify methylated DNA bound with MBD1 using its fluorescence signature.…”

mentioning

confidence: 99%

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

Cipriany

Murphy

Hagarman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing. I n chromatin, chemical modifications to histone proteins and DNA alter the status of the epigenome and influence gene regulation and normal development. Their aberrant placement has been linked to the onset of cancer (1, 2) and other diseases. Bisulfite conversion and immunoprecipitation (IP) have been used extensively to examine these modifications on locus-specific and genome-wide scales. These approaches have limitations in terms of material handling or multiplexed detection. Conventional chromatin immunoprecipitation (ChIP) requires an abundance of input material, often 10 3 -10 6 cells for genome-wide studies, to compensate for >99% material loss during processing (3, 4). This problem compounds for sequential re-ChIP reactions, limiting the study of multivalent modifications (4), which could provide a clear view of epigenetic coordination. Whereas DNA methylation analysis using bisulfite conversion can operate on picogram quantities of DNA (5-7), the conversion causes degradation of >90% of the input DNA. Methods that combine ChIP and bisulfite sequencing in a sequential process (8) have demonstrated progress in multiplexed epigenetic analysis. There continues to be active research in reducing the input material requirements and in automation of the processes (9-11) for epigenetic analysis. Furthermore, there is interest in additional capability for simultaneous detection of multiple epigenetic modifications in the same material.Miniaturized fluidic devices offer a compelling toolset for multiplexed detection and efficient sample handling in analytical and preparatory systems. Microfluidics have performed complicated workflows that include nanoliter sample handling (12) and incorporate electrodes (13-16) or valves (12, 17) for sophisticated processing. Nanofluidics have achieved attoliter-scale fluid volume confinement to isolate and quantify the attributes of individual molecules that can ...

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Engineering a high-affinity methyl-CpG-binding protein

Cited by 78 publications

References 27 publications

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions

The Three Methyl-CpG-binding Domains of AtMBD7 Control Its Subnuclear Localization and Mobility

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

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