Extraction, Identification, and Quantification of Histones from Small Quantities of Specific Brain Tissue

Hassiba Beldjoud1, Fany Messanvi1, Nael Nadif Kasri2, Benno Roozendaal3

1 Department of Neuroscience, Section Anatomy, University Medical Center Groningen, Groningen, 2 Department of Human Genetics, Radboud University Medical Center, Nijmegen, 3 Donders Institute for Brain, Cognition, and Behaviour, Nijmegen
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.38
DOI:  10.1002/cpns.11
Online Posting Date:  July, 2016
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Abstract

Histone posttranslational modifications (PTMs), by their action on the chromatin state, play a central role in the regulation of gene expression. The discovery that some PTMs in the brain are dynamically regulated by experience and environmental factors makes them an important subject for the study of plasticity changes in learning and memory, addiction, and psychiatric disorders. Current histone isolation protocols, however, require large amounts of tissue, which limits their application for analyzing small tissue samples from a specific brain region. We describe here a step‐by‐step protocol for histone extraction and isolation from 1 mm3 of tissue from brain punches, which allows reproducible and reliable results for histone PTM identification and quantification without losing anatomical precision. © 2016 by John Wiley & Sons, Inc.

Keywords: acetylation; epigenetics; histone; hippocampus; insular cortex; methylation; posttranslational modification; phosphorylation; western blotting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Brain Isolation
  • Basic Protocol 2: Slice Preparation for Microdissection
  • Basic Protocol 3: Tissue Dissection and Histone Extraction
  • Basic Protocol 4: Histone Detection by Immunoblotting (Western Blotting)
  • Support Protocol 1: Stripping PVDF Membrane
  • Support Protocol 2: Analysis of Immunoblots
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Brain Isolation

  Materials
  • Isopentane, ≥99% purity (e.g., Sigma Aldrich, cat. no. M32631)
  • Dry ice
  • Rodent of interest
  • Anesthetic (e.g., pentobarbital)
  • 250‐ml beaker
  • Thermometer capable of accurate measurements below −50°C
  • Vials or aluminum foil, for brain storage
  • Rodent guillotine
  • Surgical instruments, for brain removal
  • Round aluminum dishes with tabs
  • Forceps

Basic Protocol 2: Slice Preparation for Microdissection

  Materials
  • Frozen brain ( protocol 1)
  • Freezing mounting medium (e.g., Leica Biosystems, FSC 22 Frozen Section Media)
  • Dry ice
  • Cryostat or freezing microtome
  • Paint brushes
  • Standard glass slides
  • Razor blade
  • Forceps

Basic Protocol 3: Tissue Dissection and Histone Extraction

  Materials
  • Hypotonic lysis buffer (see recipe)
  • Tissue to be dissected (see protocol 2)
  • 0.2 N HCl
  • Trichloroacetic acid (TCA) precipitation solution (see recipe)
  • Acidified acetone: 100% acetone containing 0.1% (v/v) HCl
  • 100% acetone
  • 50 mM Tris⋅Cl (pH 8.0)/3% (w/v) SDS
  • Detergent‐compatible colorimetric assay kit (e.g., Bio‐Rad DC Protein Assay kit)
  • 5× Laemmli sample buffer (see recipe)
  • 50 mM Tris⋅Cl, pH 8.0
  • 4°C microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • 10‐ or 15‐cm petri dish
  • Palkovits punch technique brain punch set (0.75 to 1.25 mm; Stoelting, cat. no. 57401)
  • Dry ice
  • Rodent brain atlas
  • Tissue grinding pestle that fits 1.5‐ml microcentrifuge tubes (e.g., Sigma Aldrich, cat. no. Z359947‐100EA)
  • Vortex
  • Spectrophotometer
  • Heating block
CAUTION: Several of the reagents are highly corrosive (HCl, TCA, acetone). It is recommended to employ appropriate safety procedures (e.g., wearing gloves and protective clothing and working under a fume hood) and to follow the safety instructions on the manufacturers' data sheets.NOTE: All steps should be performed on ice, and all solutions and centrifuges should be chilled to 4°C prior to use.

Basic Protocol 4: Histone Detection by Immunoblotting (Western Blotting)

  Materials
  • Histone samples (see protocol 3)
  • Discontinuous polyacrylamide gel
  • 100% methanol
  • Transfer buffer (see recipe)
  • LI‐COR blocking buffer
  • PBS (see recipe)
  • TBS (see recipe)
  • 5% (w/v) non‐fat milk
  • Appropriate primary and secondary antibodies
  • Vortex
  • Microcentrifuge
  • PVDF membrane
  • Forceps
  • Laboratory shaker
  • Odyssey IR Imaging System (LI‐COR Biosciences)
  • Additional reagents and equipment for gel electrophoresis (Gallagher, ) and transfer of immunoblots (unit 5.19)
NOTE: All equipment must be clean and rinsed with water prior to use. Only critical steps are mentioned in this protocol. For a more detailed description of the immunoblotting protocol see unit 5.19 (Gallagher et al., ).

Support Protocol 1: Stripping PVDF Membrane

  Additional Materials (also see protocol 4)
  • Stripping buffer (see recipe)
  • PBS (see recipe) with and without 0.1% (v/v) Tween 20

Support Protocol 2: Analysis of Immunoblots

  Materials
  • Computer with LI‐COR Image Studio Software (available at https://www.licor.com/bio/products/software/)
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Figures

Videos

Literature Cited

Literature Cited
  Bannister, A.J. and Kouzarides, T. 2011. Regulation of chromatin by histone modifications. Cell Res. 21:381‐395. doi: 10.1038/cr.2011.22.
  Beldjoud, H., Barsegyan, A., and Roozendaal, B. 2015. Noradrenergic activation of the basolateral amygdala enhances object recognition memory and induces chromatin remodeling in the insular cortex. Front. Behav. Neurosci. 9:108. doi: 10.3389/fnbeh.2015.00108.
  Benevento, M., van de Molengraftm, M., van Westen, R., van Bokhoven, H., and Kasri, N.N. 2015. The role of chromatin repressive marks in cognition and disease: A focus on the repressive complex GLP/G9a. Neurobiol. Learn. Mem. 124:88‐96. doi: 10.1016/j.nlm.2015.06.013.
  Bousiges, O., Vasconcelos, A.P de., Neidl, R., Cosquer, B., Herbeaux, K., Panteleeva, I., Loeffler, J‐P., Cassel, J‐C., and Boutillier, A‐L. 2010. Spatial memory consolidation is associated with induction of several lysine‐acetyltransferase (histone acetyltransferase) expression levels and H2B/H4 acetylation‐dependent transcriptional events in the rat hippocampus. Neuropsychopharmacology 35:2521‐2537. doi: 10.1038/npp.2010.117.
  Cheung P., Tanner K.G., Cheung, W.L., Sassone‐Corsi, P., Denu, J.M., and Allis, C.D. 2000. Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol. Cell 5:905‐915. doi: 10.1016/S1097‐2765(00)80256‐7.
  Cohen, I., Poręba, E., Kamieniarz, K., and Schneider, R. 2011. Histone modifiers in cancer: Friends or foes? Genes Cancer 2:631‐647. doi: 10.1177/1947601911417176.
  Di Cerbo, V. and Schneider, R. 2013. Cancers with wrong HATs: The impact of acetylation. Brief. Funct. Genomics 12:231‐243. doi: 10.1093/bfgp/els065.
  Feng, J., Fouse, S., and Fan, G. 2007. Epigenetic regulation of neural gene expression and neuronal function. Pediatr. Res. 61:58R‐63R. doi: 10.1203/pdr.0b013e3180457635.
  Fischer, A., Sananbenesi, F., Wang, X., Dobbin, M, and Tsai, L‐H. 2007. Recovery of learning and memory is associated with chromatin remodelling. Nature 447:178‐182. doi: 10.1038/nature05772.
  Fischle, W., Wang, Y., and Allis, C.D. 2003. Histone and chromatin cross‐talk. Curr. Opin. Cell Biol. 15:172‐183. doi: 10.1016/S0955‐0674(03)00013‐9.
  Gallagher, S. R. 2012. One‐dimensional SDS Gel Electrophoresis of Proteins. Curr. Protoc. Prot. Sci. 68:10.1.1‐10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2004. Immunoblotting and immunodetection. Curr. Protoc. Neurosci. 29:5.19.1‐5.19.24. doi: 10.1002/0471142301.ns0519s29.
  Gräff, J. and Tsai, L‐H. 2013. Histone acetylation: Molecular mnemonics on the chromatin. Nat. Rev. Neurosci. 14:97‐111. doi: 10.1038/nrn3427.
  Gräff, J., Woldemichael, B.T., Berchtold, D., Dewarrat, G., and Mansuy, I.M. 2012. Dynamic histone marks in the hippocampus and cortex facilitate memory consolidation. Nat. Commun. 3:991. doi: 10.1038/ncomms1997.
  Gupta, S., Kim, S.Y., Artis, S., Molfese, D.L., Schumacher, A., Sweatt, J.D., Paylor, R.E., and Lubin, F.D. 2010. Histone methylation regulates memory formation. J. Neurosci. 30:3589‐3599. doi: 10.1523/JNEUROSCI.3732‐09.2010.
  Kornberg, R.D. 1974. Chromatin structure: A repeating unit of histones and DNA. Science 184:868‐871. doi: 10.1126/science.184.4139.868.
  Korzus, E., Rosenfeld, M.G., and Mayford, M. 2004. CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42:961‐972. doi: 10.1016/j.neuron.2004.06.002.
  Koshibu, K., Gräff, J., Beullens, M., Heitz, F.D., Berchtold, D., Russig, H., Farinelli, M., Bollen, M., and Mansuy, I.M. 2009. Protein phosphatase 1 regulates the histone code for long‐term memory. J. Neurosci. 29:13079‐13089. doi: 10.1523/JNEUROSCI.3610‐09.2009.
  Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128:693‐705. doi: 10.1016/j.cell.2007.02.005.
  LaSalle, J.M., Powell, W.T., and Yasui, D.H. 2013. Epigenetic layers and players underlying neurodevelopment. Trends Neurosci. 36:460‐470. doi: 10.1016/j.tins.2013.05.001.
  Levenson, J.M., O'Riordan, K.J., Brown, K.D., Trinh, M.A., Molfese, D.L., and Sweatt, J.D. 2004. Regulation of histone acetylation during memory formation in the hippocampus. J. Biol. Chem. 279:40545‐40559. doi: 10.1074/jbc.M402229200.
  Lu, L., Airey, D.C., and Williams, R.W. 2001. Complex trait analysis of the hippocampus: Mapping and biometric analysis of two novel gene loci with specific effects on hippocampal structure in mice. J. Neurosci. 21:3503‐3514.
  Martin, K.C. and Sun, Y.E. 2004. To learn better, keep the HAT on. Neuron 42:879‐881. doi: 10.1016/j.neuron.2004.06.007.
  Maze, I. and Nestler, E.J. 2011. The epigenetic landscape of addiction. Ann. N.Y. Acad. Sci. 1216:99‐113. doi: 10.1111/j.1749‐6632.2010.05893.x.
  Palkovits, M. 1973. Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59:449‐450. doi: 10.1016/0006‐8993(73)90290‐4.
  Rodriguez‐Collazo, P., Leuba, S.H., and Zlatanova, J. 2009. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications. Nucl. Acids Res 37:e81. doi: 10.1093/nar/gkp273.
  Roozendaal, B., Hernandez, A., Cabrera, S.M., Hagewoud, R., Malvaez, M., Stefanko, D.P., Haettig, J., and Wood, M.A. 2010. Membrane‐associated glucocorticoid activity is necessary for modulation of long‐term memory via chromatin modification. J. Neurosci. 30:5037‐5046. doi: 10.1523/JNEUROSCI.5717‐09.2010.
  Rudenko, A. and Tsai, L.H. 2014. Epigenetic regulation in memory and cognitive disorders. Neuroscience 264:51‐63. doi: 10.1016/j.neuroscience.2012.12.034.
  Rumbaugh, G. and Miller, C.A. 2011. Epigenetic changes in the brain: Measuring global histone modifications. Methods Mol. Biol. 670:263‐274. doi: 10.1007/978‐1‐60761‐744‐0_18.
  Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. 2007. Extraction, purification and analysis of histones. Nat. Protoc. 2:1445‐1457. doi: 10.1038/nprot.2007.202.
  Strahl, B.D. and Allis, C.D. 2000. The language of covalent histone modifications. Nature 403:41‐45. doi: 10.1038/47412.
  Tamaru, H. 2010. Confining euchromatin/heterochromatin territory: Jumonji crosses the line. Genes Dev. 24:1465‐1478. doi: 10.1101/gad.1941010.
  Tsankova, N., Renthal, W., Kumar, A., and Nestler, E.J. 2007. Epigenetic regulation in psychiatric disorders. Nat. Rev. Neurosci. 8:355‐367. doi: 10.1038/nrn2132.
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