Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.
Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.
Rodriguez-Collazo
Pedro
P
Department of Cell Biology and Physiology, Hillman Cancer Center, University of Pittsburgh, PA 15213, USA. pedrorc@pitt.edu
Leuba
Sanford H
SH
Zlatanova
Jordanka
J
D000107:Acetylation; D000818:Animals; D002460:Cell Line; D005591:Chemical Fractionation / Q000379:methods*; D002847:Chromatography, Agarose; D006657:Histones / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008938:Mitosis; D018832:Molecular Chaperones / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012685:Sepharose / Q000031:analogs & derivatives; D012965:Sodium Chloride / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D006657:Histones; D018832:Molecular Chaperones; D009707:Nucleosomes; D012965:Sodium Chloride; C035872:thiopropyl-sepharose; D014508:Urea; D012685:Sepharose