, Volume 9, Issue 6, pp 455–465 | Cite as

Age- and sex-related analysis of methylation of 5′-upstream sequences of Fmr-1 gene in mouse brain and modulation by sex steroid hormones

  • Kanchan Singh
  • S. PrasadEmail author
Research Article


Fmr-1 gene is implicated in synaptic plasticity and thereby learning, memory and cognition, and methylation of Fmr-1 gene is necessary for memory development that is an age-dependent phenomenon. Aging in general has been reported to affect methylation of gene, however, nothing is known on the age dependent variation in methylation of Fmr-1 gene. Using the brain tissues from male and female mice of various age groups and sex steroid hormones (testosterone or 17β-estradiol) as modulators, restriction enzymes Hpa II and Msp I and Southern blotting technique, we studied methylation of 5′-upstream sequences of Fmr-1 gene. Our data reveal that the methylation of the 5′-upstream sequences that include CpG islands in promoter and 5′-untraslated region (5′-UTR) gradually increases due to advancing age in both the sexes. 17β-estradiol lowers the methylation significantly in the brain of mouse of both male and female mouse in age-dependent manner where as testosterone does not affect it appreciably. The alteration in the methylation may be attributed to altered DNA methyl transferase (DNMT) activity as the age increases from young to old, and the 17β-estradiol may down regulate the DNMT activity in both the age and sex groups whereas the testosterone may not have similar effect on DNMT. Down regulation of methylation of Fmr-1 CpG island and/or 5′-UTR by 17β-estradiol might lead to derepression of Fmr-1 gene especially in old age. This finding on Fmr-1 methylation is novel and it might have implications in understanding fragile X related disorders and age-dependent alteration in LTP and LTD.


Aging Brain Aging DNA methylation Fmr-1 promoter Sex steroid hormones 



The authors acknowledge Prof. B Oostra for providing plasmid pE5.1, Profs M.S. Kanungo and M.K. Thakur, India for extending support. KS acknowledges Junior and Senior research fellowship from the UGC, Govt. of India-sponsored CAS Program in Zoology.


  1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188. doi: 10.1038/13810 PubMedCrossRefGoogle Scholar
  2. Arnold A, Gorski R (1984) Gonadal steroid induction of structural sex differences in the central nervous system. Annu Rev Neurosci 7:413–442. doi: 10.1146/ PubMedCrossRefGoogle Scholar
  3. Astolfi P, Bellizzi D, Losso MA, Sgaramella V (2001) Triplet repeats, over-expanded in neuromuscular diseases, are under-represented in mammalian DNA: a survey of models. Brain Res Bull 56:265–271. doi: 10.1016/S0361-9230(01)00581-0 PubMedCrossRefGoogle Scholar
  4. Bardoni B, Davidovic L, Bensaid M, Khandjian EW (2006) The fragile X syndrome: exploring its molecular basis and seeking a treatment. Expert Rev Mol Med 8:1–16PubMedCrossRefGoogle Scholar
  5. Brake WG, Alves SE, Dunlop JC, Lee SJ, Bulloch K, Allen PB et al (2001) Novel target sites for estrogen action in the dorsal hippocampus: an examination of synaptic proteins. Endocrinology 142:1284–1289. doi: 10.1210/en.142.3.1284 PubMedCrossRefGoogle Scholar
  6. Burak WE Jr, Quinn AL, Farrar WB, Brueggemeier RW (1997) Androgens influence estrogen-induced responses in human breast carcinoma cells through cytochrome P450 aromatase. Breast Cancer Res Treat 44:57–64. doi: 10.1023/A:1005782311558 PubMedCrossRefGoogle Scholar
  7. Chiurazzi P, Pomponi MG, Pietrobono R, Bakker CE, Neri G, Oostra BA (1999) Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene. Hum Mol Genet 8:2317–2323. doi: 10.1093/hmg/8.12.2317 PubMedCrossRefGoogle Scholar
  8. Chwang WB, O’Riordan KJ, Levenson JM, Sweatt JD (2006) ERK/ MAPK regulates hippocampal histone phosphorylation following contextual fear conditioning. Learn Mem 13:322–328. doi: 10.1101/lm.152906 PubMedCrossRefGoogle Scholar
  9. Compagnone NA, Mellon SH (2000) Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol 21:1–56. doi: 10.1006/frne.1999.0188 PubMedCrossRefGoogle Scholar
  10. Drouin R, Angers M, Dallaire N, Rose TM, Khandjian W, Rousseau F (1997) Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region. Hum Mol Genet 6:2051–2060. doi: 10.1093/hmg/6.12.2051 PubMedCrossRefGoogle Scholar
  11. Eichler EE, Richards S, Gibbs RA, Nelson D (1993) Fine structure of human FMR1 gene. Hum Mol Genet 2:1147–1153. doi: 10.1093/hmg/2.8.1147 PubMedCrossRefGoogle Scholar
  12. Fu YH, Kuhl DPA, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S et al (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67:1047–1058. doi: 10.1016/0092-8674(91)90283-5 PubMedCrossRefGoogle Scholar
  13. Garber KB, Visootsak J, Warren ST (2008) Fragile X syndrome. Eur J Hum Genet 16:666–672PubMedCrossRefGoogle Scholar
  14. Garcia-Segura LM, Inigo A, Lydia L, Don Carlos LL (2001) Neuroprotection by estradiol. Prog Neurobiol 63:29–60. doi: 10.1016/S0301-0082(00)00025-3 PubMedCrossRefGoogle Scholar
  15. Genazzani AR, Pluchino N, Luisi S, Luisi M (2007) Estrogen, cognition and female ageing. Hum Reprod Update 13:175–187. doi: 10.1093/humupd/dml042 PubMedCrossRefGoogle Scholar
  16. Genoux D, Haditsch U, Knobloch M, Michalon A, Storm D, Mansuy IM (2002) Protein phosphatase 1 is a molecular constraint on learning and memory. Nature 418:970–975. doi: 10.1038/nature00928 PubMedCrossRefGoogle Scholar
  17. Gould E, Woolley CS, Frankfurt M, McEwen BS (1990) Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J Neurosci 10:1286–1291PubMedGoogle Scholar
  18. Greenough WT, Klintsova AY, Irwin SA, Galvez R, Bates KE et al (2001) Synaptic regulation of protein synthesis and the fragile X protein. Proc Natl Acad Sci USA 98:7101–7106. doi: 10.1073/pnas.141145998 PubMedCrossRefGoogle Scholar
  19. Hansen RS, Gartler SU, Scott CR, Chen SH, Laird CD (1992) Methylation analysis of CGG sites in the CpG island of the human FMR1 gene. Hum Mol Genet 1:571–578. doi: 10.1093/hmg/1.8.571 PubMedCrossRefGoogle Scholar
  20. Hsu JC (1996) In multiple comparisons, Eds. Chapman and Hall/CRC, New YorkGoogle Scholar
  21. Huber KM, Gallagher SM, Warren ST, Bear MF (2002) Altered synaptic plasticity in a mouse model of fragile X mental retardation. Proc Natl Acad Sci USA 28:7746–7750. doi: 10.1073/pnas.122205699 CrossRefGoogle Scholar
  22. Jouvenceau A, Hedou G, Potier B, Kollen M, Dutar P, Mansuy IM (2006) Partial inhibition of PP1 alters bidirectional synaptic plasticity in the hippocampus. Eur J NeuroSci 24:564–572. doi: 10.1111/j.1460-9568.2006.04938.x PubMedCrossRefGoogle Scholar
  23. Kennesn A, Zhang F, Hagedorn CH, Warren ST (2001) Reduced FMRP and increased FMR1 transcription is proportionally associated with CGG repeat number in intermediate length and permutation carriers. Hum Mol Genet 10:1449–1454. doi: 10.1093/hmg/10.14.1449 CrossRefGoogle Scholar
  24. Keshet I, Hurwitz JL, Cedar H (1986) DNA methylation affects the formation of active chromatin. Cell 44:535–543. doi: 10.1016/0092-8674(86)90263-1 PubMedCrossRefGoogle Scholar
  25. Kumar RC, Thakur MK (2004) Androgen receptor mRNA is inversely regulated by testosterone and estradiol in adult mouse brain. Neurobiol Aging 25:925–933. doi: 10.1016/j.neurobiolaging.2003.10.011 PubMedCrossRefGoogle Scholar
  26. Kumar A, Choi KH, Renthal W, Tsankova NM, Theobold DE, Truong HT et al (2005) Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron 48:303–314. doi: 10.1016/j.neuron.2005.09.023 PubMedCrossRefGoogle Scholar
  27. Kumari D, Usdin K (2001) Interaction of the transcription factors USF1, USF2, and alpha-Pal/Nrf-1 with the FMR1 promoter: implications for fragile X mental retardation syndrome. J Biol Chem 276:4357–4364. doi: 10.1074/jbc.M009629200 PubMedCrossRefGoogle Scholar
  28. Levenson JM, Roth TL, Lubin FD, Miller CA, Huang I, Desai P et al (2006) Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 281:15763–15773. doi: 10.1074/jbc.M511767200 PubMedCrossRefGoogle Scholar
  29. Luu TT, Pirogovsky E, Gilbert PE (2008) Age-related changes in contextual associative learning. Neurobiol Learn Mem 89:81–85. doi: 10.1016/j.nlm.2008.02.005 PubMedCrossRefGoogle Scholar
  30. McEwen B (2002) Estrogen actions throughout the brain. Recent Prog Horm Res 57:357–384. doi: 10.1210/rp.57.1.357 PubMedCrossRefGoogle Scholar
  31. Miller CA, Sweatt JD (2007) Covalent modification of DNA regulates memory formation. Neuron 53:857–869. doi: 10.1016/j.neuron.2007.02.022 PubMedCrossRefGoogle Scholar
  32. Murphy DG, DeCarli C, McIntosh AR, Daly E, Mentis MJ, Pietrini P et al (1996) Sex differences in human brain morphometry and metabolism: an in vivo quantitative magnetic resonance imaging and positron emission tomography study on the effect of aging. Arch Genet Psych 53:585–594Google Scholar
  33. Norburya R, Cuttera WJ, Comptona J, Robertsona DM, Craiga M, Whiteheadb M et al (2003) The neuroprotective effects of estrogen on the aging brain. Exp Gerontol 38:109–117. doi: 10.1016/S0531-5565(02)00166-3 CrossRefGoogle Scholar
  34. Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanouer A et al (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1097–1102. doi: 10.1126/science.252.5009.1097 CrossRefGoogle Scholar
  35. O’Donnell WT, Warren ST (2002) A decade of molecular studies of fragile X syndrome. Annu Rev Neurosci 25:315–338. doi: 10.1146/annurev.neuro.25.112701.142909 PubMedCrossRefGoogle Scholar
  36. Oostra BA, Willemsen R (2002) The X chromosome and fragile X mental retardation. Cytogenet Genome Res 99:257–264. doi: 10.1159/000071602 PubMedCrossRefGoogle Scholar
  37. Penagarikano O, Mulle JG, Warren ST (2007) The pathophysiology of fragile X syndrome. Annu Rev Genomics Hum Genet 8:109–129. doi: 10.1146/annurev.genom.8.080706.092249 PubMedCrossRefGoogle Scholar
  38. Raiche J, Juarez RR, Pogribny P, Kovalchuk O (2004) Sex- and tissue-specific expression of maintenance and de novo DNA methyltransferases upon low dose X-irradiation in mice. Biochem Biophys Res Commun 325:39–47. doi: 10.1016/j.bbrc.2004.10.002 PubMedCrossRefGoogle Scholar
  39. Razin A (1998) CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J 17:4905–4908. doi: 10.1093/emboj/17.17.4905 PubMedCrossRefGoogle Scholar
  40. Richardson B (2003) Impact of aging on DNA methylation. Ageing Res Rev 2:245–261. doi: 10.1016/S1568-1637(03)00010-2 PubMedCrossRefGoogle Scholar
  41. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, (3rd edn), Cold-Spring-Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  42. Singh K, Prasad S (2007) Differential expression of Fmr1 mRNA and FMRP in old female mice brain. Mol Biol Rep. doi: 10.1007/s11033-007-9140-0
  43. Singh K, Gaur P, Prasad S (2007) Fragile X mental retardation (Fmr-1) gene expression is down regulated in brain of mice during aging. Mol Biol Rep 34:173–181. doi: 10.1007/s11033-006-9032-8 PubMedCrossRefGoogle Scholar
  44. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517. doi: 10.1016/S0022-2836(75)80083-0 PubMedCrossRefGoogle Scholar
  45. Sutcliffe JS, Nelson DL, Zhang F, Pieretti M, Caskey CT, Saxe D et al (1992) DNA methylation represses Fmr-1 transcription in fragile X syndrome. Hum Mol Genet 1:397–400. doi: 10.1093/hmg/1.6.397 PubMedCrossRefGoogle Scholar
  46. Todd PK, Mack KJ, Malter JS (2003) The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95. Proc Natl Acad Sci USA 100:14374–14378. doi: 10.1073/pnas.2336265100 PubMedCrossRefGoogle Scholar
  47. Tóth G, Gáspári Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10:967–981. doi: 10.1101/gr.10.7.967 PubMedCrossRefGoogle Scholar
  48. Veldic M, Caruncho HJ, Liu WS, Davis J, Satta R, Grayson DR et al (2004) DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains. Proc Natl Acad Sci USA 101:348–353. doi: 10.1073/pnas.2637013100 PubMedCrossRefGoogle Scholar
  49. Verkerk A, Pieretti M, Sutcliffe J, Fu YH, Kuhl D et al (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65:905–914. doi: 10.1016/0092-8674(91)90397-H PubMedCrossRefGoogle Scholar
  50. Westmark CJ, Malter JS (2007) FMRP mediates mGluR5-dependent translation of amyloid precursor protein. PLoS Biol 5:0001–0011CrossRefGoogle Scholar
  51. Wilks AF, Cozens PJ, Mattaj IW, Jost JP (1982) Estrogen induces a demethylation at the 5′ end region of the chicken vitellogenin gene. Proc Natl Acad Sci USA 79:4252–4255. doi: 10.1073/pnas.79.14.4252 PubMedCrossRefGoogle Scholar
  52. Willemsen R, Mientjes E, Oostra BA (2005) FXTAS: a progressive neurologic syndrome associated with fragile X premutation. Curr Neurol Neurosci Rep 5:405–410. doi: 10.1007/s11910-005-0065-5 PubMedCrossRefGoogle Scholar
  53. Wong TP (2002) Aging of the cerebral cortex. MJM 6:104–113Google Scholar
  54. Wood MA, Kaplan MP, Park A, Blanchard EJ, Oliveira AM, Lombardi TL et al (2005) Transgenic mice expressing a truncated form of CREB-binding protein (CBP) exhibit deficits in hippocampal synaptic plasticity and memory storage. Learn Mem 12:111–119. doi: 10.1101/lm.86605 PubMedCrossRefGoogle Scholar
  55. Woolley CS, McEwen BS (1992) Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J Neurosci 12:2549–2554PubMedGoogle Scholar
  56. Zalfa F, Achsel T, Bagni C (2006) mRNPs Polysomes or granules: FMRP in neuronal protein synthesis. Curr Opin Neurobiol 16:1–5. doi: 10.1016/j.conb.2006.05.010 CrossRefGoogle Scholar
  57. Zalfa F, Eleuteri B, Dickson KS, Mercaldo V, Silvia DR, di Penta A et al (2007) New function of the fragile X mental retardation protein in regulation of PSD-95 mRNA stability. Nat Neurosci 10:578–587. doi: 10.1038/nn1893 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.Biochemistry & Molecular Biology Laboratory, Department of ZoologyBanaras Hindu UniversityVaranasiIndia

Personalised recommendations