Dataset Information

Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

ABSTRACT:

Aim

DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions.

Experimental procedures

We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed biochemical studies of MBD2-NuRD.

Results

Most behavioral measures are minimally affected in Mbd2 knockout mice. In contrast to other MBDs, MBD2 shows distinct expression patterns.

Conclusion

Unlike most MBDs, MBD2 is ubiquitously expressed in all tissues examined and appears dispensable for brain functions measured in this study. We provide novel genetic tools and reveal new directions to investigate MBD2 functions in vivo.

SUBMITTER: Wood KH

PROVIDER: S-EPMC4928502 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

Wood Kathleen H KH Johnson Brian S BS Welsh Sarah A SA Lee Jun Y JY Cui Yue Y Krizman Elizabeth E Brodkin Edward S ES Blendy Julie A JA Robinson Michael B MB Bartolomei Marisa S MS Zhou Zhaolan Z

Epigenomics 20160412 4

<h4>Aim</h4>DNA methylation is recognized by methyl-CpG-binding domain (MBD) proteins. Multiple MBDs are linked to neurodevelopmental disorders in humans and mice. However, the functions of MBD2 are poorly understood. We characterized Mbd2 knockout mice and determined spatiotemporal expression of MBDs and MBD2-NuRD (nucleosome remodeling deacetylase) interactions.<h4>Experimental procedures</h4>We analyzed behavioral phenotypes, generated biotin-tagged MBD1 and MBD2 knockin mice, and performed b ...[more]

PMID: 27066839

Similar Datasets

Project description:BackgroundMost transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.ResultsHere, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double Strep-tag II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP.ConclusionThis study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.

Dataset Information

Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

Aim

Experimental procedures

Results

Conclusion

Publications

Tagging methyl-CpG-binding domain proteins reveals different spatiotemporal expression and supports distinct functions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets