title: Isolation and analysis of linker histones across cellular compartments.
abstract: Analysis of histones, especially histone H1, is severely limited by immunological reagent availability. This paper describes the application of cellular fractionation with LC-MS for profiling histones in the cytosol and upon chromatin. First, we show that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Second, we profiled the soluble linker histones throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. Finally, we monitored histone H1.2-H1.5 translocation to the cytosol in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows that all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell.
  
  
  CONCLUSIONS
  This paper demonstrates the first time application of cellular fractionation to characterize cytosolic histone H1 by liquid chromatography mass spectrometry (LC-MS). Using the Ramos Burkitt's lymphoma cell line, cellular fractionation was shown to give less nuclear contamination and higher histone content than preparations by nuclei isolation. Further application of the cellular fractionation approach was shown by using primary chronic lymphocytic leukemia (CLL) cells to monitor the movement of histone H1 across cellular compartments in response to the cyclin dependent kinase inhibitor flavopiridol. Collectively, these data establish a mass spectrometric method for exploration into the function of cytosolic histone H1.
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Sean W
authors.initials: SW
authors.lastname: Harshman
authors.forename: Michael M
authors.initials: MM
authors.lastname: Chen
authors.forename: Owen E
authors.initials: OE
authors.lastname: Branson
authors.forename: Naduparambil K
authors.initials: NK
authors.lastname: Jacob
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Johnson
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.forename: Michael A
authors.initials: MA
authors.lastname: Freitas
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002853:Chromatography, Liquid; D003600:Cytosol / Q000378:metabolism; D020869:Gene Expression Profiling; D015973:Gene Expression Regulation, Leukemic; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism*; D013058:Mass Spectrometry; D008938:Mitosis; D010766:Phosphorylation; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D002843:Chromatin; D006657:Histones
keywords: Cellular compartmentalization; Histone H1; LC–MS