Increase in hippocampal histone H3K9me3 is negatively correlated with memory in old male mice

  • Akanksha Kushwaha
  • Mahendra Kumar ThakurEmail author
Research Article


With advancing age, memory declines through different mechanisms including dysregulation of expression of synaptic plasticity genes in hippocampus. Increasing evidences suggest that these synaptic plasticity genes are regulated through epigenetic modifications. Recently we have reported that the neuronal immediate early genes (IEGs) are regulated by DNA methylation and histone acetylation, and their expression is downregulated in the hippocampus of old male mice, which subsequently results in decline of memory. These modifications do not work in isolation but act synergistically and lead to distinct regulation of gene expression. Therefore, in the present study, we have explored whether these genes are also regulated by histone methylation and this has any correlation with memory decline during aging. This study for the first time reports involvement of H3K9me3 in the regulation of neuronal IEGs during aging. Using novel object recognition and Y-maze test, the recognition and spatial memory was checked in male mice of different ages and it was found to decline in old. We have examined the expression of H3K9me3 specific histone methyltransferases and noted that only SUV39H1 (suppressor of variegation 3–9 homolog 1) increased significantly in old. Also the global H3K9me3 level was high in the hippocampus of old male mice. Further, chromatin immunoprecipitation assay revealed rise in H3K9me3 level at the promoter of IEGs in old as compared to young male mice. The immunofluorescence analysis also showed varying pattern of H3K9me3 expression in different subregions of hippocampus with aging. These findings showed negative correlation of increase in hippocampal histone H3K9me3 with memory decline in old male mice.

Graphic abstract

Diagram here represents that during aging, there is increase in expression of SUV39H1. Such increased enzyme upregulates global and gene specific methylation in hippocampus of old male mice. H3K9me3 level increases at the promoter of neuronal IEGs leading to heterochromatisation and hence decrease in their expression and ultimately decline in memory during aging.


Aging IEGs H3K9me3 SUV39H1 Memory Hippocampus 



The authors acknowledge the use of real-time PCR and Confocal microscope facility at the Interdisciplinary School of Life Sciences, Banaras Hindu University. Akanksha Kushwaha acknowledges Council of Scientific & Industrial Research (CSIR), India for Junior Research Fellowship. The work was financially supported by University Grants Commission, Indian Council of Medical Research (5/4-5/153/Neuro/2015-NCD-I), and Department of Science & Technology, India (EMR/2015/002178) to MKT.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Supplementary material

10522_2019_9850_MOESM1_ESM.jpg (629 kb)
Supplementary material 1—ChIP validation using anti-IgG and anti-H3K9me3 antibody (JPEG 629 kb)
10522_2019_9850_MOESM2_ESM.pdf (234 kb)
Supplementary material 2 (PDF 234 kb)


  1. Abel T, Lattal KM (2001) Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 11(2):180–187CrossRefGoogle Scholar
  2. Aenlle KK, Kumar A, Cui L et al (2009) Estrogen effects on cognition and hippocampal transcription in middle-aged mice. Neurobiol Aging 30(6):932–945CrossRefGoogle Scholar
  3. Barkus C, McHugh SB, Sprengel R et al (2010) Hippocampal NMDA receptors and anxiety: at the interface between cognition and emotion. Eur J Pharmacol 626(1):49–56. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Barski A, Cuddapah S, Cui K et al (2007) High-resolution profiling of histone methylations in the human genome. Cell 129(4):823–837CrossRefGoogle Scholar
  5. Barter JD, Foster TC (2018) Aging in the brain: new roles of epigenetics in cognitive decline. Neuroscientist 24(5):516–525. CrossRefPubMedGoogle Scholar
  6. Bilodeau S, Kagey MH, Frampton GM et al (2009) SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. Genes Dev 23(21):2484–2489. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Blalock EM, Chen KC, Sharrow K et al (2003) Gene microarrays in hippocampal aging, statistical profiling identifies novel processes correlated with cognitive impairment. J Neurosci 23(90):3807–3819CrossRefGoogle Scholar
  8. Borrelli E, Nestler EJ, Allis CD et al (2008) Decoding the epigenetic language of neuronal plasticity. Neuron 60(96):961–974. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  10. Bramham CR, Alme MN, Bittins M et al (2010) The arc of synaptic memory. Exp Brain Res 200(2):125–140. CrossRefPubMedGoogle Scholar
  11. Day JJ, Sweatt JD (2010) DNA methylation and memory formation. Nat Neurosci 13(11):1319–1323. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fahlstrom A, Yu Q, Ulfhake B (2011) Behavioral changes in aging female C57BL/6 mice. Neurobiol Aging 32(10):1868–1880. CrossRefPubMedGoogle Scholar
  13. Fuks F, Hurd PJ, Deplus R et al (2003) The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res 31(9):2305–2312CrossRefGoogle Scholar
  14. Graff J, Tsai LH (2013) Histone acetylation: molecular mnemonics on the chromatin. Nat Rev Neurosci 14(2):97–111. CrossRefPubMedGoogle Scholar
  15. Graff J, Rei D, Guan JS et al (2012) An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature 483(7388):222–226. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Greer EL, Shi Y (2012) Histone methylation: a dynamic mark in health, disease and inheritance. Nat Rev Genet 13(5):343–357. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Guan JS, Haggarty SJ, Giacometti E et al (2009) HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459(7243):55–60. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gupta S, Kim SY, Artis S et al (2010) Histone methylation regulates memory formation. J Neurosci 30(10):3589–3599. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gupta-Agarwal S, Franklin AV, Deramus T et al (2012) G9a/GLP histone lysine dimethyltransferase complex activity in the hippocampus and the entorhinal cortex is required for gene activation and silencing during memory consolidation. J Neurosci 32(16):5440–5453. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Heintzman ND, Stuart RK, Hon G et al (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 39(3):311–318CrossRefGoogle Scholar
  21. Huang PH, Chen CH, Chou CC et al (2011) Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases. Mol Pharmacol 79(1):197–206. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Ianov L, De Both M, Chawla MK et al (2017) Hippocampal transcriptomic profiles: subfield vulnerability to age and cognitive impairment. Front Aging Neurosci 9:383. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kerimoglu C, Agis-Balboa RC, Kranz A et al (2013) Histone-methyltransferase MLL2 (KMT2B) is required for memory formation in mice. J Neurosci 33(8):3452–3464. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Knapska E, Kaczmarek L (2004) A gene for neuronal plasticity in the mammalian brain: Zif268/Egr-1/NGFI-A/Krox-24/TIS8/ZENK? Prog Neurobiol 74(4):183–211CrossRefGoogle Scholar
  25. Konar A, Singh P, Thakur MK (2016) Age-associated cognitive decline, insights into molecular switches and recovery avenues. Aging Dis 7(2):121–129. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kumar A, Thakur MK (2015) Epigenetic regulation of Presenilin 1 and 2 in the cerebral cortex of mice during development. Dev Neurobiol 75(11):1165–1173. CrossRefPubMedGoogle Scholar
  27. Latham JA, Dent SY (2007) Cross-regulation of histone modifications. Nat Struct Mol Biol 14(11):1017–1024CrossRefGoogle Scholar
  28. Lee MG, Wynder C, Bochar DA et al (2006) Functional interplay between histone demethylase and deacetylase enzymes. Mol Cell Biol 26(17):6395–6402CrossRefGoogle Scholar
  29. Lein ES, Zhao X, Gage FH (2004) Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization. J Neurosci 24(15):3879–3889CrossRefGoogle Scholar
  30. Lopez-Atalaya JP, Barco A (2014) Can changes in histone acetylation contribute to memory formation? Trends Genet 30(12):529–539. CrossRefPubMedGoogle Scholar
  31. Lu T, Pan Y, Kao S et al (2004) Gene regulation and DNA damage in the aging human brain. Nature 429(6994):883–891CrossRefGoogle Scholar
  32. Lund AH, van Lohuizen M (2004) Epigenetics and cancer. Genes Dev 18(19):2315–2335CrossRefGoogle Scholar
  33. Luo P, Li X, Fei Z, Poon W (2012) Scaffold protein Homer 1: implications for neurological diseases. Neurochem Int 61(5):731–738. CrossRefPubMedGoogle Scholar
  34. Marosi K, Felszeghy K, Mehra RD (2012) Are the neuroprotective effects of estradiol and physical exercise comparable during ageing in female rats? Biogerontology 13(4):413–427. CrossRefPubMedGoogle Scholar
  35. Nightingale KP, Gendreizig S, White DA et al (2007) Cross-talk between histone modifications in response to histone deacetylase inhibitors, MLL4 links histone H3 acetylation and histone H3K4 methylation. J Biol Chem 282(7):4408–4416CrossRefGoogle Scholar
  36. O’Brien R, Xu D, Mi R et al (2002) Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons. J Neurosci 22(11):4487–4498CrossRefGoogle Scholar
  37. Palomer E, Martín-Segura A, Baliyan S et al (2016) Aging triggers a repressive chromatin state at bdnf promoters in hippocampal neurons. Cell Rep 16(11):2889–2900. CrossRefPubMedGoogle Scholar
  38. Paul CM, Magda G, Abel S (2009) Spatial memory: theoretical basis and comparative review on experimental methods in rodents. Behav Brain Res 203(2):151–164. CrossRefPubMedGoogle Scholar
  39. Peleg S, Sananbenesi F, Zovoilis A et al (2010) Altered histone acetylation is associated with age dependent memory impairment in mice. Science 328(5979):753–756. CrossRefPubMedGoogle Scholar
  40. Penner MR, Roth TL, Chawla MK et al (2011) Age-related changes in Arc transcription and DNA methylation within the hippocampus. Neurobiol Aging 32(12):2198–2210. CrossRefPubMedGoogle Scholar
  41. Peters AH, O’Carroll D, Scherthan H et al (2001) Loss of the Suv39 h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107(3):323–337CrossRefGoogle Scholar
  42. Reolon GK, Maurmann N, Werenicz A et al (2011) Posttraining systemic administration of the histone deacetylase inhibitor sodium butyrate ameliorates aging-related memory decline in rats. Behav Brain Res 221(1):329–332. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Rowe WB, Blalock EM, Chen KC et al (2007) Hippocampal expression analyses reveal selective association of immediate-early, neuroenergetic, and myelinogenic pathways with cognitive impairment in aged rats. J Neurosci 27(12):3098–3110CrossRefGoogle Scholar
  44. Schimanski LA, Barnes CA (2010) Neural protein synthesis during aging: effects on plasticity and memory. Front Aging Neurosci 2:26. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sedivy JM, Banumathy G, Adams PD (2008) Aging by epigenetics—a consequence of chromatin damage? Exp Cell Res 314(9):1909–1917. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Sims RJ 3rd, Nishioka K, Reinberg D (2003) Histone lysine methylation: a signature for chromatin function. Trends Genet 19(11):629–639CrossRefGoogle Scholar
  47. Singh P, Thakur MK (2014) Reduced recognition memory is correlated with decrease in DNA methyltransferase1 and increase in histone deacetylase2 protein expression in old male mice. Biogerontology 15(40):339–346. CrossRefPubMedGoogle Scholar
  48. Singh P, Thakur MK (2018) Histone deacetylase 2 inhibition attenuates downregulation of hippocampal plasticity gene expression during aging. Mol Neurobiol 55(3):2432–2442. CrossRefPubMedGoogle Scholar
  49. Singh P, Konar A, Kumar A et al (2015) Hippocampal chromatin modifying enzymes are pivotal for scopolamine-induced synaptic plasticity gene expression changes and memory impairment. J Neurochem 134(4):642–651. CrossRefPubMedGoogle Scholar
  50. Snigdha S, Prieto GA, Petrosyan A et al (2016) H3K9me3 inhibition improves memory, promotes spine formation, and increases BDNF levels in the aged hippocampus. J Neurosci 36(12):3611–3622. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Spencer JL, Waters EM, Romeo RD et al (2008) Uncovering the mechanisms of estrogen effects on hippocampal function. Front Neuroendocrinol 29(2):219–237CrossRefGoogle Scholar
  52. Srivas S, Thakur MK (2019) Epigenetic regulation of neuronal immediate early genes is associated with decline in their expression and memory consolidation in scopolamine-induced amnesic mice. Mol Neurobiol 56(9):6669–6672. CrossRefPubMedGoogle Scholar
  53. Stewart MD, Li J, Wong J (2005) Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment. Mol Cell Biol 25:2525–2538CrossRefGoogle Scholar
  54. Tachibana M, Sugimoto K, Nozaki M et al (2002) G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 16(14):1779–1791CrossRefGoogle Scholar
  55. Villeneuve LM, Reddy MA, Lanting LL et al (2008) Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes. Proc Natl Acad Sci USA 105(26):9047–9052. CrossRefPubMedGoogle Scholar
  56. Villeneuve LM, Kato M, Reddy MA (2010) Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1. Diabetes 59(11):2904–2915. CrossRefPubMedPubMedCentralGoogle Scholar
  57. von Ziegler LM, Selevsek N, Tweedie-Cullen RY et al (2018) Subregion-specific proteomic signature in the hippocampus for recognition processes in adult mice. Cell Rep 22(12):3362–3374. CrossRefGoogle Scholar
  58. Webb WM, Sanchez RG, Perez G et al (2017) Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory. Neurobiol Learn Mem 142(Pt A):66–78. CrossRefPubMedGoogle Scholar
  59. Wood A, Shilatifard A (2004) Posttranslational modifications of histones by methylation. Adv Protein Chem 67:201–222CrossRefGoogle Scholar
  60. Zeier Z, Madorsky I, Xu Y et al (2011) Gene expression in the hippocampus: regionally specific effects of aging and caloric restriction. Mech Ageing Dev 132(1–2):8–19. CrossRefPubMedGoogle Scholar
  61. Zhang J, He J, Chen YM et al (2008) Morphine and propranolol co-administration impair consolidation of Y-maze spatial recognition memory. Brain Res 1230:150–157. CrossRefPubMedGoogle Scholar
  62. Zhang W, Li J, Suzuki K et al (2015) Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348(6239):1160–1163. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Zheng Y, Liu A, Wang ZJ et al (2019) Inhibition of EHMT1/2 rescues synaptic and cognitive functions for Alzheimer’s disease. Brain 142(3):787–807. CrossRefPubMedGoogle Scholar
  64. Zovkic IB, Guzman-Karlsson MC, Sweatt JD (2013) Epigenetic regulation of memory formation and maintenance. Learn Mem 20(2):61–74. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

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

Personalised recommendations