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Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping.

Kernohan KD, Vernimmen D, Gloor GB, Bérubé NG - Nucleic Acids Res. (2014)

Bottom Line: ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved.Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation.We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London N6C 2V5, Canada Children's Health Research Institute, London, Canada.

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MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. (a) ATRX ChIP was performed in control and MeCP2 neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value (n = 4) and error bars depict SEM. (b) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). (c) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I (P = 0.042) of the H19 ICR and a significant decrease at site E (P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). *P < 0.05. (d) CTCF ChIP at the 5′ end of the H19 ICR (H19-2) shows decreased CTCF occupancy at this site in the MeCP2 neonatal forebrain. The graph shows mean fold change value (n = 3) and error bar depicts SEM. (e) Schematic representation of the H19/Igf2 genomic region, the position of EcoRI sites (gray vertical lines) and the primers used for 3C analysis (black arrows). Gray boxes represent the position of genes and black boxes demarcate regulatory elements. Numbers indicate the relative nucleotide position from the start of the H19 ICR. The H19 ICR bait sequence is highlighted in yellow. 3C analysis was performed with the H19 ICR bait and primers across the H19/Igf2 domain in control and MeCP2 forebrains (n = 3 littermate pairs) and was quantified by PCR with a forward primer (red arrow), Taqman probe to the H19 ICR (asterisk), and reverse primers. Graphed data represents the mean fold change of interaction frequencies, and error bars depict SEM. A two-tailed t-test was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.0001.
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Figure 4: MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. (a) ATRX ChIP was performed in control and MeCP2 neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value (n = 4) and error bars depict SEM. (b) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). (c) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I (P = 0.042) of the H19 ICR and a significant decrease at site E (P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). *P < 0.05. (d) CTCF ChIP at the 5′ end of the H19 ICR (H19-2) shows decreased CTCF occupancy at this site in the MeCP2 neonatal forebrain. The graph shows mean fold change value (n = 3) and error bar depicts SEM. (e) Schematic representation of the H19/Igf2 genomic region, the position of EcoRI sites (gray vertical lines) and the primers used for 3C analysis (black arrows). Gray boxes represent the position of genes and black boxes demarcate regulatory elements. Numbers indicate the relative nucleotide position from the start of the H19 ICR. The H19 ICR bait sequence is highlighted in yellow. 3C analysis was performed with the H19 ICR bait and primers across the H19/Igf2 domain in control and MeCP2 forebrains (n = 3 littermate pairs) and was quantified by PCR with a forward primer (red arrow), Taqman probe to the H19 ICR (asterisk), and reverse primers. Graphed data represents the mean fold change of interaction frequencies, and error bars depict SEM. A two-tailed t-test was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.0001.

Mentions: CTCF binds within an extended linker region between nucleosomes (46,47) and in vitro studies showed that irregular placement of a nucleosome within a CTCF binding site prevents the association of CTCF with that region (48). We thus speculated that ATRX, using its ATP-dependent chromatin remodeling activities, might regulate the position of nucleosomes at the maternal H19 ICR, perhaps creating a larger linker region to facilitate CTCF binding. Because ATRX and CTCF bind the maternal allele of the H19 ICR, we devised a strategy to test allele-specific nucleosome occupancy. Chromatin from control and ATRX- forebrains was digested with micrococcal nuclease and then with McrBC, an enzyme that degrades methylated DNA and should eliminate the highly methylated paternal H19 ICR (Figure 3e). The allele-specificity of this assay was validated using brain samples obtained from 129Sv/CAST polymorphic mice that have sequence differences between the paternal and maternal alleles (Figure 3f). Using this approach, we were able to compare nucleosome protection of the maternal H19 ICR in control and ATRX deficient neonatal forebrains. In the ATRX- samples, we observed increased nucleosome protection in the region of the maternal ICR corresponding to the ATRX-dependent CTCF-bound area (primer pairs B and C, Figure 4g). In the absence of ATRX, abnormal nucleosome placement is predicted to impede CTCF binding, providing a mechanistic explanation for aberrant chromosomal looping and H19/Igf2 gene expression.


Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping.

Kernohan KD, Vernimmen D, Gloor GB, Bérubé NG - Nucleic Acids Res. (2014)

MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. (a) ATRX ChIP was performed in control and MeCP2 neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value (n = 4) and error bars depict SEM. (b) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). (c) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I (P = 0.042) of the H19 ICR and a significant decrease at site E (P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). *P < 0.05. (d) CTCF ChIP at the 5′ end of the H19 ICR (H19-2) shows decreased CTCF occupancy at this site in the MeCP2 neonatal forebrain. The graph shows mean fold change value (n = 3) and error bar depicts SEM. (e) Schematic representation of the H19/Igf2 genomic region, the position of EcoRI sites (gray vertical lines) and the primers used for 3C analysis (black arrows). Gray boxes represent the position of genes and black boxes demarcate regulatory elements. Numbers indicate the relative nucleotide position from the start of the H19 ICR. The H19 ICR bait sequence is highlighted in yellow. 3C analysis was performed with the H19 ICR bait and primers across the H19/Igf2 domain in control and MeCP2 forebrains (n = 3 littermate pairs) and was quantified by PCR with a forward primer (red arrow), Taqman probe to the H19 ICR (asterisk), and reverse primers. Graphed data represents the mean fold change of interaction frequencies, and error bars depict SEM. A two-tailed t-test was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.0001.
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Figure 4: MeCP2 is required for ATRX and CTCF binding to the H19 ICR and the long-range chromatin interactions across the H19/Igf2 domain. (a) ATRX ChIP was performed in control and MeCP2 neonatal forebrain and shows that MeCP2 is required for ATRX occupancy at the H19 ICR. The graph shows mean fold change value (n = 4) and error bars depict SEM. (b) Diagram of the H19 ICR and location of the H19-1 and H19-3 primer pairs used in the ChIP-qPCR in (a). (c) Allelic nucleosome digestion assay of the H19 ICR was performed in control and MeCP2 forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2 neonatal forebrain. A significant increase in nucleosome occupancy was observed within regions B and I (P = 0.042) of the H19 ICR and a significant decrease at site E (P = 0.05). Graphs depict mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). *P < 0.05. (d) CTCF ChIP at the 5′ end of the H19 ICR (H19-2) shows decreased CTCF occupancy at this site in the MeCP2 neonatal forebrain. The graph shows mean fold change value (n = 3) and error bar depicts SEM. (e) Schematic representation of the H19/Igf2 genomic region, the position of EcoRI sites (gray vertical lines) and the primers used for 3C analysis (black arrows). Gray boxes represent the position of genes and black boxes demarcate regulatory elements. Numbers indicate the relative nucleotide position from the start of the H19 ICR. The H19 ICR bait sequence is highlighted in yellow. 3C analysis was performed with the H19 ICR bait and primers across the H19/Igf2 domain in control and MeCP2 forebrains (n = 3 littermate pairs) and was quantified by PCR with a forward primer (red arrow), Taqman probe to the H19 ICR (asterisk), and reverse primers. Graphed data represents the mean fold change of interaction frequencies, and error bars depict SEM. A two-tailed t-test was used to assess significance. *P < 0.05, **P < 0.01, ***P < 0.0001.
Mentions: CTCF binds within an extended linker region between nucleosomes (46,47) and in vitro studies showed that irregular placement of a nucleosome within a CTCF binding site prevents the association of CTCF with that region (48). We thus speculated that ATRX, using its ATP-dependent chromatin remodeling activities, might regulate the position of nucleosomes at the maternal H19 ICR, perhaps creating a larger linker region to facilitate CTCF binding. Because ATRX and CTCF bind the maternal allele of the H19 ICR, we devised a strategy to test allele-specific nucleosome occupancy. Chromatin from control and ATRX- forebrains was digested with micrococcal nuclease and then with McrBC, an enzyme that degrades methylated DNA and should eliminate the highly methylated paternal H19 ICR (Figure 3e). The allele-specificity of this assay was validated using brain samples obtained from 129Sv/CAST polymorphic mice that have sequence differences between the paternal and maternal alleles (Figure 3f). Using this approach, we were able to compare nucleosome protection of the maternal H19 ICR in control and ATRX deficient neonatal forebrains. In the ATRX- samples, we observed increased nucleosome protection in the region of the maternal ICR corresponding to the ATRX-dependent CTCF-bound area (primer pairs B and C, Figure 4g). In the absence of ATRX, abnormal nucleosome placement is predicted to impede CTCF binding, providing a mechanistic explanation for aberrant chromosomal looping and H19/Igf2 gene expression.

Bottom Line: ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved.Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation.We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London N6C 2V5, Canada Children's Health Research Institute, London, Canada.

Show MeSH
Related in: MedlinePlus