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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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Nuclear size, histone H1 composition and chromatin accessibility of wt and Mecp2tm1.1Jae mutant mice (Δ). (A) Agarose gel electrophoresis of the DNA fragments obtained by micrococcal nuclease time course digestions of mouse brain and liver at the times indicated on top of the gel. M1 and M2 are the GeneRuler 50 bp and GeneRuler 1 kb DNA ladder markers (Fermentas), respectively. The arrow points to the higher intensity of the band corresponding to the di-nucleosome in the SE fraction of the Mecp2tm1.1Jae mutant mice nuclei at high times of nuclease digestion. Bwt, BΔ and Lwt same as in Figure 4F. D: di-nucleosome; M: mono-nucleosome; T: tri-nucleosome. The numbers under the lanes shown in SE indicate the ratios D: M as determined by densitometry. (B) FRAP of wtMeCP2-GFP and ΔMeCP2-GFP.
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gkt213-F6: Nuclear size, histone H1 composition and chromatin accessibility of wt and Mecp2tm1.1Jae mutant mice (Δ). (A) Agarose gel electrophoresis of the DNA fragments obtained by micrococcal nuclease time course digestions of mouse brain and liver at the times indicated on top of the gel. M1 and M2 are the GeneRuler 50 bp and GeneRuler 1 kb DNA ladder markers (Fermentas), respectively. The arrow points to the higher intensity of the band corresponding to the di-nucleosome in the SE fraction of the Mecp2tm1.1Jae mutant mice nuclei at high times of nuclease digestion. Bwt, BΔ and Lwt same as in Figure 4F. D: di-nucleosome; M: mono-nucleosome; T: tri-nucleosome. The numbers under the lanes shown in SE indicate the ratios D: M as determined by densitometry. (B) FRAP of wtMeCP2-GFP and ΔMeCP2-GFP.

Mentions: A time course of micrococcal nuclease digestion of cell nuclei (Figure 6A) showed that chromatin from Mecp2tm1.1Jae mutant brain tissue digested much faster than chromatin from wt brains, but at a similar rate as wt liver nuclei. Interestingly, a di-nucleosome–‘resistant’ particle is observed at high-digestion times (Figure 6A, SE, 24 and 32 min), as it was also observed in Figure 5C, SE. Although the differences in the micrococcal nuclease digestion rates could be attributed to the differences in the amount of MeCP2 present in the different cell types (being the lowest in the wt liver), the amount of histone H1 was almost identical in all of them (Figure 4F). It is possible that the slower rate of digestion observed in wt brain cell nuclei is the result of a higher MeCP2 content in these cells, which is associated with a preference for binding the linker regions connecting adjacent nucleosomes (17,20). The preference of wt MeCP2 for linker DNA in proximity to the nucleosome, similar to histone H1, is likely the result of the highest levels of DNA methylation within these chromatin regions (59) and the affinity of MeCP2 for four-way junction DNA (60), a structure that resembles the DNA organization at its site of entry and exit from the nucleosome. Accordingly, histone H1, which also binds at this nucleosome site, exhibits a similar preference for four-way junctions (61).Figure 6.


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)

Nuclear size, histone H1 composition and chromatin accessibility of wt and Mecp2tm1.1Jae mutant mice (Δ). (A) Agarose gel electrophoresis of the DNA fragments obtained by micrococcal nuclease time course digestions of mouse brain and liver at the times indicated on top of the gel. M1 and M2 are the GeneRuler 50 bp and GeneRuler 1 kb DNA ladder markers (Fermentas), respectively. The arrow points to the higher intensity of the band corresponding to the di-nucleosome in the SE fraction of the Mecp2tm1.1Jae mutant mice nuclei at high times of nuclease digestion. Bwt, BΔ and Lwt same as in Figure 4F. D: di-nucleosome; M: mono-nucleosome; T: tri-nucleosome. The numbers under the lanes shown in SE indicate the ratios D: M as determined by densitometry. (B) FRAP of wtMeCP2-GFP and ΔMeCP2-GFP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt213-F6: Nuclear size, histone H1 composition and chromatin accessibility of wt and Mecp2tm1.1Jae mutant mice (Δ). (A) Agarose gel electrophoresis of the DNA fragments obtained by micrococcal nuclease time course digestions of mouse brain and liver at the times indicated on top of the gel. M1 and M2 are the GeneRuler 50 bp and GeneRuler 1 kb DNA ladder markers (Fermentas), respectively. The arrow points to the higher intensity of the band corresponding to the di-nucleosome in the SE fraction of the Mecp2tm1.1Jae mutant mice nuclei at high times of nuclease digestion. Bwt, BΔ and Lwt same as in Figure 4F. D: di-nucleosome; M: mono-nucleosome; T: tri-nucleosome. The numbers under the lanes shown in SE indicate the ratios D: M as determined by densitometry. (B) FRAP of wtMeCP2-GFP and ΔMeCP2-GFP.
Mentions: A time course of micrococcal nuclease digestion of cell nuclei (Figure 6A) showed that chromatin from Mecp2tm1.1Jae mutant brain tissue digested much faster than chromatin from wt brains, but at a similar rate as wt liver nuclei. Interestingly, a di-nucleosome–‘resistant’ particle is observed at high-digestion times (Figure 6A, SE, 24 and 32 min), as it was also observed in Figure 5C, SE. Although the differences in the micrococcal nuclease digestion rates could be attributed to the differences in the amount of MeCP2 present in the different cell types (being the lowest in the wt liver), the amount of histone H1 was almost identical in all of them (Figure 4F). It is possible that the slower rate of digestion observed in wt brain cell nuclei is the result of a higher MeCP2 content in these cells, which is associated with a preference for binding the linker regions connecting adjacent nucleosomes (17,20). The preference of wt MeCP2 for linker DNA in proximity to the nucleosome, similar to histone H1, is likely the result of the highest levels of DNA methylation within these chromatin regions (59) and the affinity of MeCP2 for four-way junction DNA (60), a structure that resembles the DNA organization at its site of entry and exit from the nucleosome. Accordingly, histone H1, which also binds at this nucleosome site, exhibits a similar preference for four-way junctions (61).Figure 6.

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