Molecular Dissection of Chromatin Maturation via Click Chemistry

Ozlem Yildirim1, Robert E. Kingston1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.33
DOI:  10.1002/0471142727.mb2133s114
Online Posting Date:  April, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

DNA synthesis and chromatin assembly are the two most critical processes of eukaryotic cell division. It is well known that their coordination is tightly regulated. Although the interplay between DNA and its higher‐order chromatin state is integral for many processes, including cell survival and genome stability, little is known about the re‐establishment of chromatin structure during the cell cycle. Moreover, the extent to which the fidelity of the newly synthesized chromatin plays a role in the maintenance of cellular identity is still under debate. Here, we present a novel approach to purify nascent chromatin from the replication fork. In this protocol, we take advantage of click chemistry, a method that allows efficient conjugation of azide‐containing biotin molecules to ethynyl‐labeled nucleic acids. Using this approach, we selectively enrich biotin‐nucleic acid conjugates via streptavidin affinity purification to pull down and assess chromatin states as well as chromatin‐bound complexes from newly replicated DNA fragments. © 2016 by John Wiley & Sons, Inc.

Keywords: epigenetic inheritance; cell cycle; nascent chromatin; EdU; click chemistry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Nascent Chromatin with Click Chemistry
  • Support Protocol 1: Assessing Click Efficiency with DNA Dot/Slot‐Blot
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Nascent Chromatin with Click Chemistry

  Materials
  • Cultured human telomerase reverse transcriptase (hTERT) immortalized RPE1 cells (ATCC CRL‐4000; 8 × 106 to 10 × 106 cells for each experiment)
  • RPE1 medium (see recipe)
  • 5‐Ethynyl‐2′‐deoxyuridine (EdU; Berry & Associates, cat. no. PY7562; or 5‐ethynyl‐2′‐deoxycytidine [EdC; Jena Bioscience, cat. no. CLK‐N003])
  • L‐Mimosine (Sigma‐Aldrich, cat. no. M0253)
  • Mimosine stock solution (see recipe)
  • Nuclease‐free water (Ambion, cat. no. AM9932)
  • 100% ethanol
  • Formaldehyde (Thermo Fisher Scientific, cat. no.28908)
  • Tween 20 (Sigma‐Aldrich, cat. no. P1379)
  • Glycine (Sigma‐Aldrich, cat. no. 50046)
  • Triton X‐100 (Sigma‐Aldrich, cat. no. T93443)
  • Glycogen (Ambion, cat. no. AM9510)
  • Dynabeads MyOne Streptavidin C1 magnetic beads (Life Technologies, cat. no. 65001)
  • 3 M sodium acetate, pH 5.5 (Ambion, cat. no. AM9740)
  • Mammalian protease inhibitor cocktail (Sigma‐Aldrich, cat. no. P8340)
  • RNasin Plus Rnase inhibitor (Thermo Fisher Scientific, cat. no. PRN2615)
  • 10% SDS (Life Technologies, cat. no. AM 9822)
  • Proteinase K (Life Technologies, cat. no. AM2548)
  • 50× Denhardt's blocking buffer (Life Technologies, cat. no. 750018)
  • 2× Tween 20 wash buffer (see recipe)
  • Click reaction mixture (see recipe)
  • Cytoplasm wash buffer (see recipe)
  • DTT wash buffer (see recipe)
  • Pull‐down sonication buffer (see recipe)
  • TE buffer, nuclease free (Life Technologies, cat. no. AM9858)
  • RIPA buffer (see recipe)
  • 5× Reducing sample buffer (Thermo Fisher Scientific, cat. no. 39000)
  • RNAse A (Qiagen, cat. no. 19101)
  • Nuclease‐free BSA (Sigma‐Aldrich, cat. no. B2518)
  • Dyna Mag magnetic stand (Life Technologies, cat. no. 12321D)
  • QSonica, sonicator (QSonica, cat. no. Q800R1)
  • Cell lifter (Corning, cat. no. 3008)
  • Low‐binding nuclease‐free 1.7‐ml tubes (Eppendorf, cat. no. 022431021)
  • Thermo mixer (Eppendorf, cat. no. 022670107)
  • Rotator (Thomas Scientific, cat. no. 1217H25)
  • 1.5‐ml Qsonica sonication tubes
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA (unit 2.1, Moore and Dowhan, ) and agarose gel electrophoresis (unit 2.5, Voytas, )
NOTE: Prepare all solutions with nuclease‐free water.

Support Protocol 1: Assessing Click Efficiency with DNA Dot/Slot‐Blot

  Additional Materials (also see protocol 1Basic Protocol)
  • Ambion 20× SSC (Thermo Fisher Scientific, cat. no. AM9763)
  • 10% bleach
  • TE buffer (optional)
  • 1 M NaOH
  • 0.5 M EDTA
  • Tris‐buffered saline and Tween 20 (TBST)
  • Pierce high sensitivity streptavidin‐HRP, pre‐diluted (Thermo Fisher Scientific, cat. no. 21134)
  • Nucleic acid detection blocking buffer (Thermo Fisher Scientific, cat. no. 89880 A)
  • Ammonium acetate (Sigma‐Aldrich, cat. no. 09689)
  • Streptavidin‐HRP wash buffer (see recipe)
  • Biodyne B nylon membrane (Thermo Fisher Scientific cat. no. 77016,)
  • Whatman 3 MM paper
  • Bio‐Dot apparatus (Bio‐Rad)
  • UV crosslinker (UV Stratalinker 2400)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Alabert, C., Bukowski‐Wills, J.‐C., Lee, S.‐B., Kustatscher, G., Nakamura, K., de Lima Alves, F., Menard, P., Mejlvang, J., Rappsilber, J., and Groth, A. 2014. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat. Cell Biol. 16:281‐293. doi: 10.1038/ncb2918.
  Brown, T. 1999. Dot and slot blotting of DNA. Curr. Protoc. Mol. Biol. 21:2.9.15‐2.9.20.
  Budhavarapu, V.N., Chavez, M., and Tyler, J.K. 2013. How is epigenetic information maintained through DNA replication? Epigenetics Chromatin 6:32. doi: 10.1186/1756‐8935‐6‐32.
  Gramlich, P.M.E., Warncke, S., Gierlich, J., and Carell, T. 2008. Click‐Click‐Click: Single to triple modification of DNA. Angew. Chem. Int. Ed. 47:3442‐3444. doi: 10.1002/anie.200705664.
  Groth, A. 2009. Replicating chromatin: A tale of histones. Biochem. Cell Biol. 87:51‐63. doi: 10.1139/O08‐102.
  Henikoff, S., Furuyama, T., and Ahmad, K. 2004. Histone variants, nucleosome assembly and epigenetic inheritance. Trends Genet. 20:320‐326. doi: 10.1016/j.tig.2004.05.004.
  Kliszczak, A.E.A., Rainey, M.D.M., Harhen, B.B., Boisvert, F.M.F., and Santocanale, C.C. 2011. DNA mediated chromatin pull‐down for the study of chromatin replication. Sci. Rep. 1:95. doi: 10.1038/srep00095.
  Kolb, H.C., Finn, M.G., and Sharpless, K.B. 2001. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40:2004‐2021. doi: 10.1002/1521‐3773(20010601)40:11%3c2004::AID‐ANIE2004%3e3.0.CO;2‐5.
  Marzluff, W.F., Wagner, E.J., and Duronio, R.J. 2008. Metabolism and regulation of canonical histone mRNAs: Life without a poly(A) tail. Nat. Rev. Genet. 9:843‐854. doi: 10.1038/nrg2438.
  Mejlvang, J., Feng, Y., Alabert, C., Neelsen, K.J., Jasencakova, Z., Zhao, X., Lees, M., Sandelin, A., Pasero, P., Lopes, M., and Groth, A. 2014. New histone supply regulates replication fork speed and PCNA unloading. J. Cell Biol. 204:29‐43. doi: 10.1083/jcb.201305017.
  Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1‐2.1.10. doi: 10.1002/0471142727.mb0201as59.
  Paredes, E. and Das, S.R. 2010. Click chemistry for rapid labeling and ligation of RNA. ChemBioChem 12:125‐131. doi: 10.1002/cbic.201000466.
  Rando, O.J. 2007. Global patterns of histone modifications. Curr. Opin. Genet. Dev. 17:94‐99. doi: 10.1016/j.gde.2007.02.006.
  Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise Huisgen cycloaddition process: Copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41:2596‐2599. doi: 10.1002/1521‐3773(20020715)41:14%3c2596::AID‐ANIE2596%3e3.0.CO;2‐4.
  Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415‐2420. doi: 10.1073/pnas.0712168105.
  Sirbu, B.M.B., Couch, F.B.F., Feigerle, J.T.J., Bhaskara, S.S., Hiebert, S.W.S., and Cortez, D.D. 2011. Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev. 25:1320‐1327. doi: 10.1101/gad.2053211.
  Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9. doi: 10.1002/0471142727.mb0205as51.
  Ye, X., Franco, A.A., Santos, H., Nelson, D.M., Kaufman, P.D., and Adams, P.D. 2003. Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest. Mol. Cell 11:341‐351. doi: 10.1016/S1097‐2765(03)00037‐6.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library