Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping.
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.
Affiliation: Department of Biochemistry, University of Western Ontario, London N6C 2V5, Canada Children's Health Research Institute, London, Canada.Show MeSH
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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.
Affiliation: Department of Biochemistry, University of Western Ontario, London N6C 2V5, Canada Children's Health Research Institute, London, Canada.