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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.

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Distribution of wtMeCP2 and ΔMeCP2 in different sections of the brain. The upper and lower part show a western blot analysis using an MeCP2 and histone H3 antibody, respectively, of the SDS–PAGE analysis of the nuclear protein composition shown in the middle. CB: cerebellum; CT: cortex and MB: midbrain.
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gkt213-F2: Distribution of wtMeCP2 and ΔMeCP2 in different sections of the brain. The upper and lower part show a western blot analysis using an MeCP2 and histone H3 antibody, respectively, of the SDS–PAGE analysis of the nuclear protein composition shown in the middle. CB: cerebellum; CT: cortex and MB: midbrain.

Mentions: Wild-type MeCP2 has been shown to be differentially expressed in various brain regions, with the highest expression in cerebellum and cortex (48–50). Therefore, and because of the reduced expression of the protein, we decided to check its expression in different regions of the brain. As seen in Figure 2, ΔMeCP2 is expressed throughout different sections of the brain. However, although a noticeable increase in the expression of wtMeCP2 was detected in wt cortex as previously described, no significant difference could be observed for the expression of ΔMeCP2 among different regions of the Mecp2tm1.1Jae mutant brain.Figure 2.


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)

Distribution of wtMeCP2 and ΔMeCP2 in different sections of the brain. The upper and lower part show a western blot analysis using an MeCP2 and histone H3 antibody, respectively, of the SDS–PAGE analysis of the nuclear protein composition shown in the middle. CB: cerebellum; CT: cortex and MB: midbrain.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt213-F2: Distribution of wtMeCP2 and ΔMeCP2 in different sections of the brain. The upper and lower part show a western blot analysis using an MeCP2 and histone H3 antibody, respectively, of the SDS–PAGE analysis of the nuclear protein composition shown in the middle. CB: cerebellum; CT: cortex and MB: midbrain.
Mentions: Wild-type MeCP2 has been shown to be differentially expressed in various brain regions, with the highest expression in cerebellum and cortex (48–50). Therefore, and because of the reduced expression of the protein, we decided to check its expression in different regions of the brain. As seen in Figure 2, ΔMeCP2 is expressed throughout different sections of the brain. However, although a noticeable increase in the expression of wtMeCP2 was detected in wt cortex as previously described, no significant difference could be observed for the expression of ΔMeCP2 among different regions of the Mecp2tm1.1Jae mutant brain.Figure 2.

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