Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin

  • Eustermann S
  • Yang J
  • Law M
 et al. 
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The chromatin-associated human protein ATRX was originally iden-tified because mutations in the ATRX gene cause a severe form of syn-dromal X-linked mental retardation called ATR-X syndrome 1 . This syndrome is associated with profound developmental delay, facial dysmorphism, urogenital abnormalities and α-thalassemia 2 . Somatic mutations are found in the pre-leukemic condition α-thalassemia myelodysplastic syndrome 3 , and more recently, ATRX has also been identified as a potential tumor-suppressor gene in pancreatic neuro-endocrine tumors 4 . ATRX syndrome mutations or knockdown of ATRX expression cause diverse effects, including altered patterns of DNA methyla-tion 5 , a telomere-dysfunction phenotype 6 , aberrant chromosome segregation 7 , premature sister chromatid separation 8 and changes in gene expression 1,9 . ATRX localizes predominantly to large, tandemly repeated regions (such as telomeres, centromeres and ribosomal DNA) associated with heterochromatin 9–11 , and recent evidence shows that it directs H3.3 deposition to pericentric and telomeric hetero-chromatin 10,12 . In addition, we have previously shown that ATRX is enriched at chromatin sites that contain tandemly repeated GC-rich DNA sequences, where it may be involved in modifying G-quadruplex DNA structures 3 . However, the mechanism of ATRX recruitment to heterochromatin remains largely unknown. Targeting of ATRX to heterochromatin requires H3 tails bearing trimethylated Lys9 (H3K9me3); when the Suv39H1 and Suv39H2 methyltransferases that add these methyl groups are knocked out, ATRX no longer localizes to pericentric heterochromatin (see ref. 13 and Supplementary Fig. 1a). This observation was originally rationalized by proposing an indirect mechanism 11 , whereby ATRX interacts with the C-terminal chromo shadow domains of HP1α and HP1β proteins 14,15 that in turn recognize H3K9me3 by means of their N-terminal chromodomains 16 . However, such a mechanism would provide little specificity, as HP1 is a widely distributed, constitutive component of heterochromatin that also interacts with many other proteins 14 . We therefore considered whether ATRX might itself inter-act with histone tails directly. Our previous structural analysis of the ADD domain of ATRX 17 revealed that it contains a PHD zinc-finger domain packed against a GATA-like zinc finger (Fig. 1), a structure that was subsequently also found in the DNMT3 DNA methyltrans-ferases and DNMT3L 18,19 . Given the relevance of the ADD domain for ATRX function, as evidenced by the large number of syndrome-causing mutations in this domain (Fig. 1), and the known role of PHD zinc fingers as H3 histone recognition modules, we hypothesized that the ADD domain has a direct function in ATRX chromatin recruitment. RESULTS Both the ADD domain and HP1 contribute to ATRX recruitment In order to assess the relative contributions of the ADD domain and the HP1 interaction to chromatin recruitment of ATRX, we intro-duced point mutations in these two regions (singly or in combina-tion) and analyzed the localization of ATRX in vivo. To determine the role of the ADD domain, we tested a construct containing a disease-causing mutation (C240G) that eliminates a zinc-binding cysteine in the PHD zinc finger and is known to destabilize the structure of the ADD domain 17 . The role of the 'Leu-X-Val-X-Leu' HP1 interaction

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  • Sebastian Eustermann

  • Ji Chun Yang

  • Martin J. Law

  • Rachel Amos

  • Lynda M. Chapman

  • Clare Jelinska

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