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

Abstract

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 show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. 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. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure.

Figure 1.

4C-sequencing analysis of chromosome interactions of the H19 ICR bait sequence in neonatal mouse forebrain. (a) 3C libraries were generated from neonatal forebrain utilizing EcoRI, then re-digested with MseI and self-ligated to form circular 3C recombined molecules. The samples were then amplified with primers directed from the H19 ICR ‘bait sequence’ across the interacting fragments and sequenced. Venn diagrams show the number of common sequences between 4C-seq biological replicates. Interactions of the H19 ICR in trans are represented on the left and interactions in cis are represented on the right. (b) Analysis of genomic distribution of H19 ICR interacting fragments on each chromosome reveals that the majority of reproducible interactions occur within chromosome 7 while trans interactions are distributed across the genome. (c) Representative 4C interaction profile across chromosome 7 (top) and the H19/Igf2 imprinted domain (bottom). Local interactions are observe with the Igf2 DMR1, MAR3, CCD and downstream enhancers. The 4C-seq data was aligned to an EcoRI digested genome and the H19 ICR bait sequence is highlighted in yellow.

Figure 2.

3C analysis of control and ATRX-deficient neonatal forebrain shows that ATRX is required for long-range chromosomal interactions mediated by the H19 ICR. (a) Analysis of ATRX ChIP-sequencing data in mouse embryonic stem cells (15) shows ATRX occupancy at several imprinted domains (left panels, UCSC views). ATRX enrichment at these sites in neonatal mouse forebrain was confirmed by ChIP, as shown in the graphs on the left (n = 3, error bars represent SEM, p = peak, adj = adjacent). (b) 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 ATRX-null forebrains (n = 5 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.

Figure 3.

ATRX regulates nucleosome occupancy and CTCF binding at the H19 ICR. (a) Schematic representation of the H19 ICR and alignment of primers used for ChIP-qPCR (top) and nucleosome occupancy analysis (bottom). Gray boxes indicate the positions of CTCF binding sites and a red box marks the ATRX-binding site. Numbers indicate the relative position from the start of the H19 ICR. (b) ChIP of ATRX in E13.5 and P0.5 forebrains shows binding of ATRX in the middle portion of the H19 ICR (H19-3) at P0.5, as observed previously, but not at E13.5, indicating that ATRX is recruited to the H19 ICR in the late embryonic/neonatal brain. (c) CTCF ChIP at E13.5 and P0.5 shows binding of CTCF at both time points at previously identified areas of occupancy (H19-2 and H19-4). (d) ChIP of CTCF in control and ATRX-null forebrain tissue at E13.5 and P0.5 reveals that ATRX binding is required for CTCF binding at P0.5, but not at E13.5. Graphs in (b), (c) and (d) represent mean values, n = 3 for each and error bars depict SEM. (e) Diagram depicting the methodology used for allele-specific micrococcal nuclease digestion. Empty circles indicate unmethylated CpGs and black circles indicate methylated CpGs. (f) Validation of allele-specificity of nucleosome occupancy protocol, in which the methylated paternal H19 ICR sequence is digested by McrBC. In F1 polymorphic 129Sv (maternal)/castaneous (paternal) forebrain samples, MfeI digests 129Sv maternal DNA and McrBC digests methylated paternal DNA. Following digestion, DNA was amplified using primers spanning the MfeI restriction site. (g) qPCR of micrococcal nuclease and McrBC-digested DNA reveals increased protection at the 5′ end of the maternal H19 ICR in the ATRX-null samples. A significant increase in nucleosome occupancy was observed at regions B (P = 0.016) and C (P = 0.05) of the H19 ICR and a significant decrease at adjacent site G (P = 0.001). Graph shows mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). *P < 0.05.

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^null 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^null forebrains and reveals increased protection at the 5′ end of the maternal H19 ICR in the MeCP2^null 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^null 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^null 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.

Figure 5.

ATRX and MeCP2 regulate nucleosome positioning and long-range chromatin interactions mediated by the Gtl2 DMR. (a) 4C-sequencing analysis using the Gtl2 DMR as bait was performed in wild-type neonatal forebrains. Venn diagrams show the number of common sequences between 4C-seq biological replicates. Interactions of the Gtl2 DMR in trans are represented on the left and interactions in cis are represented on the right. (b) Analysis of genomic distribution of Gtl2 DMR-interacting fragments on each chromosome reveals that the majority of reproducible interactions occur within chromosome 12 while trans interactions are distributed across the genome, with a noted enrichment on the sex chromosomes. (c) Schematic representation of the Gtl2/Dlk1 genomic region, the position of EcoRI sites (black 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 Gtl2. The Gtl2 DMR bait sequence is highlighted in yellow. (d) For 3C analysis, DNA was digested with EcoRI, ligated and quantified by real-time PCR with a forward primer (red arrow) and Taqman probe to the Gtl2 DMR (asterisk), and reverse primers (black arrows). Analysis was performed in control and ATRX-null or control and MeCP2-null neonatal forebrains (n = 3 littermate matched pairs each). A significant reduction in interaction frequency is observed at specific sites including the Dlk1 gene and many intergenic regions. Graphed data represents the mean fold change, and error bars depict SEM. A two-tailed t-test was used to assess significance. *P < 0.05, **p < 0.01, ***p < 0.0001. (e) qPCR of micrococcal nuclease and McrBC digested DNA reveals increased DNA protection in the ATRX-null (left) MeCP2-null (right) forebrain overlapping the CTCF binding site in the Gtl2 DMR. Graphs depict mean fold change and statistical analysis was performed by a two-tailed t-test (n = 3, errors bars depict SEM). **P < 0.01.

Figure 6.

Model of ATRX and MeCP2 function. (a) In the wild-type brain, MeCP2 recruits ATRX to the maternal H19 ICR in the late embryonic/neonatal period. ATRX translocates along the chromatin fiber and alters nucleosome positioning to generate an extended linker region and promote CTCF occupancy. CTCF then dictates intrachromosomal interactions. (b) In the absence of ATRX, increased nucleosome occupancy disrupts CTCF binding, leading to a loss of intrachromosomal interactions.