Limits...
Impaired in vivo binding of MeCP2 to chromatin in the absence of its DNA methyl-binding domain.

Stuss DP, Cheema M, Ng MK, Martinez de Paz A, Williamson B, Missiaen K, Cosman JD, McPhee D, Esteller M, Hendzel M, Delaney K, Ausió J - Nucleic Acids Res. (2013)

Bottom Line: However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood.Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak.Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Victoria, British Columbia, V8W 2Y2, Canada.

ABSTRACT
MeCP2 is a methyl-CpG-binding protein that is a main component of brain chromatin in vertebrates. In vitro studies have determined that in addition to its specific methyl-CpG-binding domain (MBD) MeCP2 also has several chromatin association domains. However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood. We analysed the role of the MBD in MeCP2-chromatin associations in vivo using an MeCP2 mutant Rett syndrome mouse model (Mecp2(tm1.1Jae)) in which exon 3 deletion results in an N-terminal truncation of the protein, including most of the MBD. Our results show that in mutant mice, the truncated form of MeCP2 (ΔMeCP2) is expressed in different regions of the brain and liver, albeit at 50% of its wild-type (wt) counterpart. In contrast to the punctate nuclear distribution characteristic of wt MeCP2, ΔMeCP2 exhibits both diffuse nuclear localization and a substantial retention in the cytoplasm, suggesting a dysfunction of nuclear transport. In mutant brain tissue, neuronal nuclei are smaller, and ΔMeCP2 chromatin is digested faster by nucleases, producing a characteristic nuclease-resistant dinucleosome. Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak. Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

Show MeSH

Related in: MedlinePlus

ΔMeCP2 has altered nuclear distribution in fixed brain tissue and is present in the cytoplasm. (A) Confocal fluorescence images of MeCP2-immunolabelled coronal sections of motor cortex layer 5. Top eight panels (males): Nuclei are counter-stained with YO-PRO-1 (blue). Neuronal nuclei are larger, typically with bright punctate staining corresponding to dense heterochromatin, whereas glial nuclei are smaller and sometimes irregularly shaped. In wt (Mecp2+/y) males, both α-N-terminal (green) and α-C-terminal (red) MeCP2 immunolabelling show bright puncta against a diffuse nuclear background. In MeCP2J mutant (Mecp2-/y) males, α-N-terminal MeCP2 labelling is absent, whereas faint diffuse nuclear α-C-terminal MeCP2 labelling is still observed, consistent with the N-terminal deletion and absence of the MBD. Glial MeCP2 signal is not evident in either genotype. Scale bar = 40 µm. Bottom six (female): Heterozygous YFP+Mecp2+/− females have a mixture of both MeCP2 immunolabelling patterns (α-N-terminal, blue; α-C-terminal, red). α-C-terminal-MeCP2 labelling is comparatively faint in mutant cells, but can be seen in a high-exposure image (bottom left, greyscale). Under high exposure, immunolabelling in wt neurons for both antibodies is bright to oversaturated, but it remains exclusively nuclear (arrow #3). In mutant neurons, faint diffuse α-C-terminal MeCP2 immunolabelling is predominantly nuclear, but it also occurs in the cytoplasm extending into dendrites (arrows #1, #2). Scale bar = 40 µm. (B) SDS–PAGE (1) and western blot (2) analysis of overall MeCP2 present in wt and mutant brain in comparison with the cytoplasmic (C) and nuclear (N) fractions. The red asterisk indicates a degradation product of wt MeCP2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643609&req=5

gkt213-F3: ΔMeCP2 has altered nuclear distribution in fixed brain tissue and is present in the cytoplasm. (A) Confocal fluorescence images of MeCP2-immunolabelled coronal sections of motor cortex layer 5. Top eight panels (males): Nuclei are counter-stained with YO-PRO-1 (blue). Neuronal nuclei are larger, typically with bright punctate staining corresponding to dense heterochromatin, whereas glial nuclei are smaller and sometimes irregularly shaped. In wt (Mecp2+/y) males, both α-N-terminal (green) and α-C-terminal (red) MeCP2 immunolabelling show bright puncta against a diffuse nuclear background. In MeCP2J mutant (Mecp2-/y) males, α-N-terminal MeCP2 labelling is absent, whereas faint diffuse nuclear α-C-terminal MeCP2 labelling is still observed, consistent with the N-terminal deletion and absence of the MBD. Glial MeCP2 signal is not evident in either genotype. Scale bar = 40 µm. Bottom six (female): Heterozygous YFP+Mecp2+/− females have a mixture of both MeCP2 immunolabelling patterns (α-N-terminal, blue; α-C-terminal, red). α-C-terminal-MeCP2 labelling is comparatively faint in mutant cells, but can be seen in a high-exposure image (bottom left, greyscale). Under high exposure, immunolabelling in wt neurons for both antibodies is bright to oversaturated, but it remains exclusively nuclear (arrow #3). In mutant neurons, faint diffuse α-C-terminal MeCP2 immunolabelling is predominantly nuclear, but it also occurs in the cytoplasm extending into dendrites (arrows #1, #2). Scale bar = 40 µm. (B) SDS–PAGE (1) and western blot (2) analysis of overall MeCP2 present in wt and mutant brain in comparison with the cytoplasmic (C) and nuclear (N) fractions. The red asterisk indicates a degradation product of wt MeCP2.

Mentions: Previous immunohistochemical data from the Mecp2tm1.1Jae line have been contradictory, with the original study showing a loss of immunoreactivity in histological sections of hippocampus and cerebellum [see (29), Figure 1F], while a subsequent study found diffuse ΔMeCP2 staining in the hippocampus [see (31), Figure 2G]. We re-investigated this issue by double-immunolabelling cortical brain slices using the mouse N-terminal α-MeCP2 and a chicken C-terminal α-MeCP2 to compare wt males, mutant males and heterozygous Mecp2+/− females (Figure 3A). We found that successful double-immunolabelling was highly sensitive to tissue fixation conditions, which may account for the discrepant results in previous reports. The immunoreactivity of the C-terminal antibody was robust under most fixation conditions, but the N-terminal antibody immunoreactivity was extremely sensitive to overfixation, and was only reliably obtained after a brief formaldehyde fixation using a pH-shift protocol (53). In wt males, both antibodies labelled all neurons with a typical staining pattern showing bright puncta against a diffuse nuclear background (Figure 3A, top). Immunolabelled puncta co-localized with heterochromatin regions, as previously described (3,54). In hemizygous Mecp2−/y mutant males, only the C-terminal antibody was immunoreactive, and nuclear labelling was faint and diffuse, with no puncta observed (Figure 3A, bottom). To facilitate comparison of the subcellular distribution patterns of wt with mutant MeCP2 in vivo, heterozygous females were obtained from a cross of the Mecp2tm1.1Jae line with the B6.Cg-Tg(Thy1-YFPH)2Jrs/J line, which express YFP in a subset of cortical neurons. Heterozygous Mecp2+/− females showed a mixture of the two patterns of immunoreactivity observed in males. When imaged under high-exposure conditions to amplify the fluorescence signal, wt MeCP2 labelled with both antibodies was strictly localized to the nucleus. In neurons labelled by the C-terminal antibody alone, immunoreactivity was also observed in the cytoplasm, extending up into the primary apical dendrite, albeit at much lower levels. The marked difference in nuclear staining at the border of the nucleus relative to adjacent cytoplasm indicated that ΔMeCP2 still retained a predominantly nuclear localization.Figure 3.


Impaired in vivo binding of MeCP2 to chromatin in the absence of its DNA methyl-binding domain.

Stuss DP, Cheema M, Ng MK, Martinez de Paz A, Williamson B, Missiaen K, Cosman JD, McPhee D, Esteller M, Hendzel M, Delaney K, Ausió J - Nucleic Acids Res. (2013)

ΔMeCP2 has altered nuclear distribution in fixed brain tissue and is present in the cytoplasm. (A) Confocal fluorescence images of MeCP2-immunolabelled coronal sections of motor cortex layer 5. Top eight panels (males): Nuclei are counter-stained with YO-PRO-1 (blue). Neuronal nuclei are larger, typically with bright punctate staining corresponding to dense heterochromatin, whereas glial nuclei are smaller and sometimes irregularly shaped. In wt (Mecp2+/y) males, both α-N-terminal (green) and α-C-terminal (red) MeCP2 immunolabelling show bright puncta against a diffuse nuclear background. In MeCP2J mutant (Mecp2-/y) males, α-N-terminal MeCP2 labelling is absent, whereas faint diffuse nuclear α-C-terminal MeCP2 labelling is still observed, consistent with the N-terminal deletion and absence of the MBD. Glial MeCP2 signal is not evident in either genotype. Scale bar = 40 µm. Bottom six (female): Heterozygous YFP+Mecp2+/− females have a mixture of both MeCP2 immunolabelling patterns (α-N-terminal, blue; α-C-terminal, red). α-C-terminal-MeCP2 labelling is comparatively faint in mutant cells, but can be seen in a high-exposure image (bottom left, greyscale). Under high exposure, immunolabelling in wt neurons for both antibodies is bright to oversaturated, but it remains exclusively nuclear (arrow #3). In mutant neurons, faint diffuse α-C-terminal MeCP2 immunolabelling is predominantly nuclear, but it also occurs in the cytoplasm extending into dendrites (arrows #1, #2). Scale bar = 40 µm. (B) SDS–PAGE (1) and western blot (2) analysis of overall MeCP2 present in wt and mutant brain in comparison with the cytoplasmic (C) and nuclear (N) fractions. The red asterisk indicates a degradation product of wt MeCP2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643609&req=5

gkt213-F3: ΔMeCP2 has altered nuclear distribution in fixed brain tissue and is present in the cytoplasm. (A) Confocal fluorescence images of MeCP2-immunolabelled coronal sections of motor cortex layer 5. Top eight panels (males): Nuclei are counter-stained with YO-PRO-1 (blue). Neuronal nuclei are larger, typically with bright punctate staining corresponding to dense heterochromatin, whereas glial nuclei are smaller and sometimes irregularly shaped. In wt (Mecp2+/y) males, both α-N-terminal (green) and α-C-terminal (red) MeCP2 immunolabelling show bright puncta against a diffuse nuclear background. In MeCP2J mutant (Mecp2-/y) males, α-N-terminal MeCP2 labelling is absent, whereas faint diffuse nuclear α-C-terminal MeCP2 labelling is still observed, consistent with the N-terminal deletion and absence of the MBD. Glial MeCP2 signal is not evident in either genotype. Scale bar = 40 µm. Bottom six (female): Heterozygous YFP+Mecp2+/− females have a mixture of both MeCP2 immunolabelling patterns (α-N-terminal, blue; α-C-terminal, red). α-C-terminal-MeCP2 labelling is comparatively faint in mutant cells, but can be seen in a high-exposure image (bottom left, greyscale). Under high exposure, immunolabelling in wt neurons for both antibodies is bright to oversaturated, but it remains exclusively nuclear (arrow #3). In mutant neurons, faint diffuse α-C-terminal MeCP2 immunolabelling is predominantly nuclear, but it also occurs in the cytoplasm extending into dendrites (arrows #1, #2). Scale bar = 40 µm. (B) SDS–PAGE (1) and western blot (2) analysis of overall MeCP2 present in wt and mutant brain in comparison with the cytoplasmic (C) and nuclear (N) fractions. The red asterisk indicates a degradation product of wt MeCP2.
Mentions: Previous immunohistochemical data from the Mecp2tm1.1Jae line have been contradictory, with the original study showing a loss of immunoreactivity in histological sections of hippocampus and cerebellum [see (29), Figure 1F], while a subsequent study found diffuse ΔMeCP2 staining in the hippocampus [see (31), Figure 2G]. We re-investigated this issue by double-immunolabelling cortical brain slices using the mouse N-terminal α-MeCP2 and a chicken C-terminal α-MeCP2 to compare wt males, mutant males and heterozygous Mecp2+/− females (Figure 3A). We found that successful double-immunolabelling was highly sensitive to tissue fixation conditions, which may account for the discrepant results in previous reports. The immunoreactivity of the C-terminal antibody was robust under most fixation conditions, but the N-terminal antibody immunoreactivity was extremely sensitive to overfixation, and was only reliably obtained after a brief formaldehyde fixation using a pH-shift protocol (53). In wt males, both antibodies labelled all neurons with a typical staining pattern showing bright puncta against a diffuse nuclear background (Figure 3A, top). Immunolabelled puncta co-localized with heterochromatin regions, as previously described (3,54). In hemizygous Mecp2−/y mutant males, only the C-terminal antibody was immunoreactive, and nuclear labelling was faint and diffuse, with no puncta observed (Figure 3A, bottom). To facilitate comparison of the subcellular distribution patterns of wt with mutant MeCP2 in vivo, heterozygous females were obtained from a cross of the Mecp2tm1.1Jae line with the B6.Cg-Tg(Thy1-YFPH)2Jrs/J line, which express YFP in a subset of cortical neurons. Heterozygous Mecp2+/− females showed a mixture of the two patterns of immunoreactivity observed in males. When imaged under high-exposure conditions to amplify the fluorescence signal, wt MeCP2 labelled with both antibodies was strictly localized to the nucleus. In neurons labelled by the C-terminal antibody alone, immunoreactivity was also observed in the cytoplasm, extending up into the primary apical dendrite, albeit at much lower levels. The marked difference in nuclear staining at the border of the nucleus relative to adjacent cytoplasm indicated that ΔMeCP2 still retained a predominantly nuclear localization.Figure 3.

Bottom Line: However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood.Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak.Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Victoria, British Columbia, V8W 2Y2, Canada.

ABSTRACT
MeCP2 is a methyl-CpG-binding protein that is a main component of brain chromatin in vertebrates. In vitro studies have determined that in addition to its specific methyl-CpG-binding domain (MBD) MeCP2 also has several chromatin association domains. However, the specific interactions of MeCP2 with methylated or non-methylated chromatin regions and the structural characteristics of the resulting DNA associations in vivo remain poorly understood. We analysed the role of the MBD in MeCP2-chromatin associations in vivo using an MeCP2 mutant Rett syndrome mouse model (Mecp2(tm1.1Jae)) in which exon 3 deletion results in an N-terminal truncation of the protein, including most of the MBD. Our results show that in mutant mice, the truncated form of MeCP2 (ΔMeCP2) is expressed in different regions of the brain and liver, albeit at 50% of its wild-type (wt) counterpart. In contrast to the punctate nuclear distribution characteristic of wt MeCP2, ΔMeCP2 exhibits both diffuse nuclear localization and a substantial retention in the cytoplasm, suggesting a dysfunction of nuclear transport. In mutant brain tissue, neuronal nuclei are smaller, and ΔMeCP2 chromatin is digested faster by nucleases, producing a characteristic nuclease-resistant dinucleosome. Although a fraction of ΔMeCP2 is found associated with nucleosomes, its interaction with chromatin is transient and weak. Thus, our results unequivocally demonstrate that in vivo the MBD of MeCP2 together with its adjacent region in the N-terminal domain are critical for the proper interaction of the protein with chromatin, which cannot be replaced by any other of its protein domains.

Show MeSH
Related in: MedlinePlus