Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Targeting G-quadruplex DNA as cognitive function therapy for ATR-X syndrome

Abstract

Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome is caused by mutations in ATRX, which encodes a chromatin-remodeling protein. Genome-wide analyses in mouse and human cells indicate that ATRX tends to bind to G-rich sequences with a high potential to form G-quadruplexes. Here, we report that Atrx mutation induces aberrant upregulation of Xlr3b expression in the mouse brain, an outcome associated with neuronal pathogenesis displayed by ATR-X model mice. We show that ATRX normally binds to G-quadruplexes in CpG islands of the imprinted Xlr3b gene, regulating its expression by recruiting DNA methyltransferases. Xlr3b binds to dendritic mRNAs, and its overexpression inhibits dendritic transport of the mRNA encoding CaMKII-α, promoting synaptic dysfunction. Notably, treatment with 5-ALA, which is converted into G-quadruplex-binding metabolites, reduces RNA polymerase II recruitment and represses Xlr3b transcription in ATR-X model mice. 5-ALA treatment also rescues decreased synaptic plasticity and cognitive deficits seen in ATR-X model mice. Our findings suggest a potential therapeutic strategy to target G-quadruplexes and decrease cognitive impairment associated with ATR-X syndrome.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ATRX regulates Xlr3b gene expression.
Fig. 2: Xlr3b is found in neuronal RNA granules.
Fig. 3: Aberrant Xlr3b expression perturbs synaptic plasticity by inhibiting dendritic Camk2a mRNA transport.
Fig. 4: 5-ALA represses Xlr3b transcription with Pol II recruitment by modifying the G-quadruplex structure.
Fig. 5: Treatment with 5-ALA counteracts cognitive deficits seen in AtrxΔE2 mice.

References

  1. Gibbons, R. J., Suthers, G. K., Wilkie, A. O., Buckle, V. J. & Higgs, D. R. X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12–q21.31 by X inactivation and linkage analysis. Am. J. Hum. Genet. 51, 1136–1149 (1992).

    PubMed  PubMed Central  CAS  Google Scholar 

  2. Gibbons, R. J., Picketts, D. J., Villard, L. & Higgs, D. R. Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell 80, 837–845 (1995).

    Article  PubMed  CAS  Google Scholar 

  3. Gibbons, R. J. et al. Mutations in the chromatin-associated protein ATRX. Hum. Mutat. 29, 796–802 (2008).

    Article  PubMed  CAS  Google Scholar 

  4. Argentaro, A. et al. Structural consequences of disease-causing mutations in the ATRX–DNMT3–DNMT3L (ADD) domain of the chromatin-associated protein ATRX. Proc. Natl Acad. Sci. USA 104, 11939–11944 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Dhayalan, A. et al. The ATRX–ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9. Hum. Mol. Genet. 20, 2195–2203 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Iwase, S. et al. ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome. Nat. Struct. Mol. Biol. 18, 769–776 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Picketts, D. J. et al. ATRX encodes a novel member of the SNF2 family of proteins: mutations point a common mechanism underlying the ATR-X syndrome. Hum. Mol. Genet. 5, 1899–1907 (1996).

    Article  PubMed  CAS  Google Scholar 

  8. Mitson, M., Kelley, L. A., Sternberg, M. J., Higgs, D. R. & Gibbons, R. J. Functional significance of mutations in the Snf2 domain of ATRX. Hum. Mol. Genet. 20, 2603–2610 (2011).

    Article  PubMed  CAS  Google Scholar 

  9. Law, M. J. et al. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell 143, 367–378 (2010).

    Article  PubMed  CAS  Google Scholar 

  10. Drané, P., Ouararhni, K., Depaux, A., Shuaib, M. & Hamiche, A. The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3. Genes Dev. 24, 1253–1265 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Goldberg, A. D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Lewis, P. W., Elsaesser, S. J., Noh, K. M., Stadler, S. C. & Allis, C. D. Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc. Natl Acad. Sci. USA 107, 14075–14080 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Voon, H. P. et al. ATRX plays a key role in maintaining silencing at interstitial heterochromatic loci and imprinted genes. Cell Rep. 11, 405–418 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Kernohan, K. D. et al. ATRX partners with cohesin and MeCP2 and contributes to developmental silencing of imprinted genes in the brain. Dev. Cell 18, 191–202 (2010).

    Article  PubMed  CAS  Google Scholar 

  15. Kernohan, K. D., Vernimmen, D., Gloor, G. B. & Bérubé, N. G. Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping. Nucleic Acids Res. 42, 8356–8368 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Levy, M. A., Kernohan, K. D., Jiang, Y. & Bérubé, N. G. ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions. Hum. Mol. Genet. 24, 1824–1835 (2015).

    Article  PubMed  CAS  Google Scholar 

  17. Butler, M. G. Genomic imprinting disorders in humans: a mini-review. J. Assist. Reprod. Genet. 26, 477–486 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Shioda, N. et al. Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain. J. Neurosci. 31, 346–358 (2011).

    Article  PubMed  CAS  Google Scholar 

  19. Nogami, T. et al. Reduced expression of the ATRX gene, a chromatin-remodeling factor, causes hippocampal dysfunction in mice. Hippocampus 21, 678–687 (2011).

    Article  PubMed  CAS  Google Scholar 

  20. Howard, M. T. et al. Attenuation of an amino-terminal premature stop codon mutation in the ATRX gene by an alternative mode of translational initiation. J. Med. Genet. 41, 951–956 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Abidi, F. E. Mutation in the 5′ alternatively spliced region of the XNP/ATR-X gene causes Chudley–Lowry syndrome. Eur. J. Hum. Genet. 13, 176–183 (2005).

    Article  PubMed  CAS  Google Scholar 

  22. Bergsagel, P. L., Timblin, C. R., Kozak, C. A. & Kuehl, W. M. Sequence and expression of murine cDNAs encoding Xlr3a and Xlr3b, defining a new X-linked lymphocyte-regulated Xlr gene subfamily. Gene 150, 345–350 (1994).

    Article  PubMed  CAS  Google Scholar 

  23. Raefski, A. S. & O’Neill, M. J. Identification of a cluster of X-linked imprinted genes in mice. Nat. Genet. 37, 620–624 (2005).

    Article  PubMed  CAS  Google Scholar 

  24. Davies, W. et al. Xlr3b is a new imprinted candidate for X-linked parent-of-origin effects on cognitive function in mice. Nat. Genet. 37, 625–629 (2005).

    Article  PubMed  CAS  Google Scholar 

  25. Moore, L. D., Le, T. & Fan, G. DNA methylation and its basic function. Neuropsychopharmacology 38, 23–38 (2013).

    Article  PubMed  CAS  Google Scholar 

  26. Hutnick, L. K. et al. DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation. Hum. Mol. Genet. 18, 2875–2888 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat. Neurosci. 13, 423–430 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Gibbons, R. J. et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat. Genet. 24, 368–371 (2000).

    Article  PubMed  CAS  Google Scholar 

  29. Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Glickman, M. H. & Ciechanover, A. The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    Article  PubMed  CAS  Google Scholar 

  31. Martinez-Garay, I. et al. A new gene family (FAM9) of low-copy repeats in Xp22.3 expressed exclusively in testis: implications for recombinations in this region. Genomics 80, 259–267 (2002).

    Article  PubMed  CAS  Google Scholar 

  32. Hirokawa, N. & Takemura, R. Molecular motors and mechanisms of directional transport in neurons. Nat. Rev. Neurosci. 6, 201–214 (2005).

    Article  PubMed  CAS  Google Scholar 

  33. Bramham, C. R. & Wells, D. G. Dendritic mRNA: transport, translation and function. Nat. Rev. Neurosci. 8, 776–789 (2007).

    Article  PubMed  CAS  Google Scholar 

  34. Rook, M. S., Lu, M. & Kosik, K. S. CaMKIIα 3′ untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage. J. Neurosci. 20, 6385–6393 (2000).

    Article  PubMed  CAS  Google Scholar 

  35. Bliss, T. V. & Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39 (1993).

    Article  PubMed  CAS  Google Scholar 

  36. Fukunaga, K., Muller, D. & Miyamoto, E. Increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in the induction of long-term potentiation. J. Biol. Chem. 270, 6119–6124 (1995).

    Article  PubMed  CAS  Google Scholar 

  37. Scheetz, A. J., Nairn, A. C. & Constantine-Paton, M. NMDA receptor-mediated control of protein synthesis at developing synapses. Nat. Neurosci. 3, 211–216 (2000).

    Article  PubMed  CAS  Google Scholar 

  38. Miller, S. et al. Disruption of dendritic translation of CaMKIIα impairs stabilization of synaptic plasticity and memory consolidation. Neuron 36, 507–519 (2002).

    Article  PubMed  CAS  Google Scholar 

  39. Fukunaga, K., Stoppini, L., Miyamoto, E. & Muller, D. Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 268, 7863–7867 (1993).

    PubMed  CAS  Google Scholar 

  40. Ouyang, Y., Kantor, D., Harris, K. M., Schuman, E. M. & Kennedy, M. B. Visualization of the distribution of autophosphorylated calcium/calmodulin-dependent protein kinase II after tetanic stimulation in the CA1 area of the hippocampus. J. Neurosci. 17, 5416–5427 (1997).

    Article  PubMed  CAS  Google Scholar 

  41. Balasubramanian, S., Hurley, L. H. & Neidle, S. Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat. Rev. Drug Discov. 10, 261–275 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Peng, Q. et al. 5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges. Cancer 79, 2282–2308 (1997).

    Article  PubMed  CAS  Google Scholar 

  43. Qin, Y. & Hurley, L. H. Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie 90, 1149–1171 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Bochman, M. L., Paeschke, K. & Zakian, V. A. DNA secondary structures: stability and function of G-quadruplex structures. Nat. Rev. Genet. 13, 770–780 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Kikin, O., Zappala, Z., D’Antonio, L. & Bagga, P. S. GRSDB2 and GRS_UTRdb: databases of quadruplex forming G-rich sequences in pre-mRNAs and mRNAs. Nucleic Acids Res. 36, D141–D148 (2008).

    Article  PubMed  CAS  Google Scholar 

  46. Gibbons, R. Alpha thalassaemia-mental retardation, X linked. Orphanet J. Rare Dis. 1, 15 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Villanueva, A. & Jori, G. Pharmacokinetic and tumour-photosensitizing properties of the cationic porphyrin meso-tetra(4N-methylpyridyl)porphine. Cancer Lett. 73, 59–64 (1993).

    Article  PubMed  CAS  Google Scholar 

  48. Dalton, J. T. et al. Clinical pharmacokinetics of 5-aminolevulinic acid in healthy volunteers and patients at high risk for recurrent bladder cancer. J. Pharmacol. Exp. Ther. 301, 507–512 (2002).

    Article  PubMed  CAS  Google Scholar 

  49. Perotti, C., Casas, A., Fukuda, H., Sacca, P. & Batlle, A. ALA and ALA hexyl ester induction of porphyrins after their systemic administration to tumour bearing mice. Br. J. Cancer 87, 790–795 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Skalska, L., Beltran-Nebot, M., Ule, J. & Jenner, R. G. Regulatory feedback from nascent RNA to chromatin and transcription. Nat. Rev. Mol. Cell Biol. 18, 331–337 (2017).

    Article  PubMed  CAS  Google Scholar 

  51. Clynes, D. & Gibbons, R. J. ATRX and the replication of structured DNA. Curr. Opin. Genet. Dev. 23, 289–294 (2013).

    Article  PubMed  CAS  Google Scholar 

  52. Li, Y. et al. Effect of ATRX and G-quadruplex formation by the VNTR sequence on α-globin gene expression. ChemBiochem 17, 928–935 (2016).

    Article  PubMed  CAS  Google Scholar 

  53. Roberts, D. W. et al. Glioblastoma multiforme treatment with clinical trials for surgical resection (aminolevulinic acid). Neurosurg. Clin. N. Am. 23, 371–377 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Stummer, W. et al. Intraoperative detection of malignant gliomas by 5-aminolevulinic acid-induced porphyrin fluorescence. Neurosurgery 42, 518–526 (1998).

    Article  PubMed  CAS  Google Scholar 

  55. Al-Saber, F. et al. The safety and tolerability of 5-aminolevulinic acid phosphate with sodium ferrous citrate in patients with type 2 diabetes mellitus in Bahrain. J. Diabetes Res. 2016, 8294805 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Shioda, N. et al. Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRβ signaling in striatopallidal medium spiny neurons. Mol. Psychiatry 22, 1205–1222 (2017).

    Article  PubMed  CAS  Google Scholar 

  57. Spencer, C. M., Alekseyenko, O., Serysheva, E., Yuva-Paylor, L. A. & Paylor, R. Altered anxiety-related and social behaviors in the Fmr1 knockout mouse model of fragile X syndrome. Genes Brain Behav. 4, 420–430 (2005).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank I. Kitajima for kindly providing AtrxΔE2 mice, deposited in the RIKEN BioResource Center (RBRC04937), H. Shimbo for kindly assisting in cell culture, D. Picketts for kindly providing the Atrx cDNA (pEGFP-C2-ATRX-HA) plasmid, N. Berube for kindly providing the ATRX shRNA (pSUPER-shATRX1) plasmid and K. Kosik for kindly providing the GFP-MS2-nls and MS2-binding site–CaMKII-α 3′ UTR plasmids. This research was supported by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development (AMED; N.S., K.K., H.T., N.O., H.S., K.F. and T.W.). This work was also supported by MEXT/JSPS KAKENHI (grant numbers 16K08265 and 25110705) to N.S.

Author information

Authors and Affiliations

Authors

Contributions

N.S., Y.Y., K.Y., M.O. and Y.L. performed the experiments. K.K., H.T., N.O., T.E., H.S. and T.W. provided critical advice. N.S. and K.F. wrote the manuscript and designed the study. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Norifumi Shioda, Takahito Wada or Kohji Fukunaga.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Tables 1–5

Reporting Summary

Supplementary Video 1

Time-lapse imaging of GFP-MS2-labeled CaMKIIα mRNA (GFP-CaMKIIα 3′ UTR) in a proximal dendrite of a cultured WT neuron

Supplementary Video 2

Time-lapse imaging of GFP-MS2-labeled CaMKIIα mRNA (GFP-CaMKIIα 3′ UTR) in a distal dendrite of a cultured WT neuron

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shioda, N., Yabuki, Y., Yamaguchi, K. et al. Targeting G-quadruplex DNA as cognitive function therapy for ATR-X syndrome. Nat Med 24, 802–813 (2018). https://doi.org/10.1038/s41591-018-0018-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41591-018-0018-6

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing