Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

Jost, K. Laurence; Rottach, Andrea; Milden, Manuela; Bertulat, Bianca; Becker, Annette; Wolf, Patricia; Sandoval, Juan; Petazzi, Paolo; Huertas, Dori; Esteller, Manel; Kremmer, Elisabeth; Leonhardt, Heinrich; Cardoso, M. Cristina

doi:10.1371/journal.pone.0026499

Cited by 19 publications

(27 citation statements)

References 30 publications

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“…The primary rabbit anti Mecp2 antibody (32) (1:2) was applied for 1 h at RT. After three washing steps using PBST containing 0.01% Tween, the secondary donkey anti rabbit IgG cy3 (1:500, The Jackson Laboratory, Bar Harbor, USA) was applied for 45 min at RT.…”

Section: Methodsmentioning

confidence: 99%

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise

Ludwig

Zhang

Hastert

et al. 2016

Nucleic Acids Research

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Aberrant DNA methylation is a hallmark of various human disorders, indicating that the spatial and temporal regulation of methylation readers and modifiers is imperative for development and differentiation. In particular, the cross-regulation between 5-methylcytosine binders (MBD) and modifiers (Tet) has not been investigated. Here, we show that binding of Mecp2 and Mbd2 to DNA protects 5-methylcytosine from Tet1-mediated oxidation. The mechanism is not based on competition for 5-methylcytosine binding but on Mecp2 and Mbd2 directly restricting Tet1 access to DNA. We demonstrate that the efficiency of this process depends on the number of bound MBDs per DNA molecule. Accordingly, we find 5-hydroxymethylcytosine enriched at heterochromatin of Mecp2-deficient neurons of a mouse model for Rett syndrome and Tet1-induced reexpression of silenced major satellite repeats. These data unveil fundamental regulatory mechanisms of Tet enzymes and their potential pathophysiological role in Rett syndrome. Importantly, it suggests that Mecp2 and Mbd2 have an essential physiological role as guardians of the epigenome.

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Section: Methodsmentioning

confidence: 99%

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise

Ludwig

Zhang

Hastert

et al. 2016

Nucleic Acids Research

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“…Specificity of the antibody was checked using fibroblasts derived from Mecp2 - /y and Mecp2 lox/y mice (Additional file 1). In some cases, rat monoclonal antibodies were used as well [65]. The antibodies for cell type identification and for recognition of retinal structures are listed in Tables 1 and 3.…”

Section: Methodsmentioning

confidence: 99%

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

Song

Feodorova

Guy

et al. 2014

Epigenetics & Chromatin

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BackgroundMethyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.ResultsUsing immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2-/y ) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2-/y and Mecp2 wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2-/y mice.ConclusionsMECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.

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“…To produce pMeCP2R.9, pMeCP2G.9 [29] was digested with BamHI and NheI releasing the MeCP2.9 fragment. The insert was then ligated into pmRFP-N2 vector, cut before with the same restriction sites.…”

Section: Methodsmentioning

confidence: 99%

“…Full length MeCP2 constructs tagged with GFP (MeCP2G) and strep (stMeCP2) were described before [29].…”

Section: Methodsmentioning

confidence: 99%

Direct Homo- and Hetero-Interactions of MeCP2 and MBD2

et al. 2013

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Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163–309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.

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Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

Cited by 19 publications

References 30 publications

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise

Binding of MBD proteins to DNA blocks Tet1 function thereby modulating transcriptional noise

DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution

Direct Homo- and Hetero-Interactions of MeCP2 and MBD2

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