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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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Expression of ΔMeCP2 in Mecp2tm1.1Jae mutant mice. (A) Schematic representation of mouse MeCP2 E1. The N-terminal domain (NTD), MBD, the ID, the C-terminal domains (2), NLS (32) and the two PEST sequences (33) are indicated. (B) SDS–PAGE and western blot of nuclear extracts from Mecp2tm1.1Jae mutant (Δ) and wt mouse brain. CM, chicken erythrocyte histone marker. Top: SDS–PAGE loadings were normalized based on histone H4 contents. Bottom: Western blots were performed using C-terminal MeCP2 (left) or N-terminal MeCP2 (right) antibodies. Both of them label a higher MW band in wt nuclear extracts but only the C-terminal antibody labels a lower MW band in the mutant (Δ), consistent with the presence of a truncated MeCP2 protein with an N-terminal deletion in the MBD. (C) RT–PCR analysis of wtMeCP2 and ΔMeCP2 expression in brain.
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gkt213-F1: Expression of ΔMeCP2 in Mecp2tm1.1Jae mutant mice. (A) Schematic representation of mouse MeCP2 E1. The N-terminal domain (NTD), MBD, the ID, the C-terminal domains (2), NLS (32) and the two PEST sequences (33) are indicated. (B) SDS–PAGE and western blot of nuclear extracts from Mecp2tm1.1Jae mutant (Δ) and wt mouse brain. CM, chicken erythrocyte histone marker. Top: SDS–PAGE loadings were normalized based on histone H4 contents. Bottom: Western blots were performed using C-terminal MeCP2 (left) or N-terminal MeCP2 (right) antibodies. Both of them label a higher MW band in wt nuclear extracts but only the C-terminal antibody labels a lower MW band in the mutant (Δ), consistent with the presence of a truncated MeCP2 protein with an N-terminal deletion in the MBD. (C) RT–PCR analysis of wtMeCP2 and ΔMeCP2 expression in brain.

Mentions: Methyl-CpG-binding protein 2 (MeCP2) is a basic chromosomal protein that binds to symmetrical methylated 5′CpG dinucleotide sequences (1). Although the protein consists of several well-defined structural domains (2) (Figure 1A), it is an excellent example of an intrinsically disordered protein (5) because of a very low content of secondary structure organization (40%) (5,6). Intrinsically disordered proteins contain low levels of secondary structure that can increase on interaction with other binding partners (7). In addition to DNA, MeCP2 has numerous protein interaction partners (8) and has been shown to interact with RNA (9). It should thus not come as a surprise that mutations throughout the whole protein have potentially deleterious consequences.Figure 1.


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)

Expression of ΔMeCP2 in Mecp2tm1.1Jae mutant mice. (A) Schematic representation of mouse MeCP2 E1. The N-terminal domain (NTD), MBD, the ID, the C-terminal domains (2), NLS (32) and the two PEST sequences (33) are indicated. (B) SDS–PAGE and western blot of nuclear extracts from Mecp2tm1.1Jae mutant (Δ) and wt mouse brain. CM, chicken erythrocyte histone marker. Top: SDS–PAGE loadings were normalized based on histone H4 contents. Bottom: Western blots were performed using C-terminal MeCP2 (left) or N-terminal MeCP2 (right) antibodies. Both of them label a higher MW band in wt nuclear extracts but only the C-terminal antibody labels a lower MW band in the mutant (Δ), consistent with the presence of a truncated MeCP2 protein with an N-terminal deletion in the MBD. (C) RT–PCR analysis of wtMeCP2 and ΔMeCP2 expression in brain.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt213-F1: Expression of ΔMeCP2 in Mecp2tm1.1Jae mutant mice. (A) Schematic representation of mouse MeCP2 E1. The N-terminal domain (NTD), MBD, the ID, the C-terminal domains (2), NLS (32) and the two PEST sequences (33) are indicated. (B) SDS–PAGE and western blot of nuclear extracts from Mecp2tm1.1Jae mutant (Δ) and wt mouse brain. CM, chicken erythrocyte histone marker. Top: SDS–PAGE loadings were normalized based on histone H4 contents. Bottom: Western blots were performed using C-terminal MeCP2 (left) or N-terminal MeCP2 (right) antibodies. Both of them label a higher MW band in wt nuclear extracts but only the C-terminal antibody labels a lower MW band in the mutant (Δ), consistent with the presence of a truncated MeCP2 protein with an N-terminal deletion in the MBD. (C) RT–PCR analysis of wtMeCP2 and ΔMeCP2 expression in brain.
Mentions: Methyl-CpG-binding protein 2 (MeCP2) is a basic chromosomal protein that binds to symmetrical methylated 5′CpG dinucleotide sequences (1). Although the protein consists of several well-defined structural domains (2) (Figure 1A), it is an excellent example of an intrinsically disordered protein (5) because of a very low content of secondary structure organization (40%) (5,6). Intrinsically disordered proteins contain low levels of secondary structure that can increase on interaction with other binding partners (7). In addition to DNA, MeCP2 has numerous protein interaction partners (8) and has been shown to interact with RNA (9). It should thus not come as a surprise that mutations throughout the whole protein have potentially deleterious consequences.Figure 1.

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