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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.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.

SUBMITTER: Harshman SW

PROVIDER: S-EPMC3863389 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Isolation and analysis of linker histones across cellular compartments.

Harshman Sean W SW Chen Michael M MM Branson Owen E OE Jacob Naduparambil K NK Johnson Amy J AJ Byrd John C JC Freitas Michael A MA

Journal of proteomics 20130905

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 phosphory ...[more]

PMID: 24013129

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Project description:H1s, or linker histones, are ubiquitous proteins in eukaryotic cells, consisting of a globular GH1 domain flanked by two unstructured tails. Whilst it is known that numerous non-allelic variants exist within the same species, the degree of interspecific and intraspecific variation and divergence of linker histones remain unknown. The conserved basic binding sites in GH1 and evenly distributed strong positive charges on the C-terminal domain (CTD) are key structural characters for linker histones to bind chromatin. Based on these features, we identified five linker histones from 13 GH1-containing proteins in castor bean (Ricinus communis), which were named as RcH1.1, RcH1.2a, RcH1.2b, RcH1.3, and RcH1.4 based on their phylogenetic relationships with the H1s from five other economically important Euphorbiaceae species (Hevea brasiliensis Jatropha curcas, Manihot esculenta Mercurialis annua, and Vernicia fordii) and Arabidopsis thaliana. The expression profiles of RcH1 genes in a variety of tissues and stresses were determined from RNA-seq data. We found three RcH1 genes (RcH1.1, RcH1.2a, and RcH1.3) were broadly expressed in all tissues, suggesting a conserved role in stabilizing and organizing the nuclear DNA. RcH1.2a and RcH1.4 was preferentially expressed in floral tissues, indicating potential involvement in floral development in castor bean. Lack of non-coding region and no expression detected in any tissue tested suggest that RcH1.2b is a pseudogene. RcH1.3 was salt stress inducible, but not induced by cold, heat and drought in our investigation. Structural comparison confirmed that GH1 domain was highly evolutionarily conserved and revealed that N- and C-terminal domains of linker histones are divergent between variants, but highly conserved between species for a given variant. Although the number of H1 genes varies between species, the number of H1 variants is relatively conserved in more closely related species (such as within the same family). Through comparison of nucleotide diversity of linker histone genes and oil-related genes, we found similar mutation rate of these two groups of genes. Using Tajima's D and ML-HKA tests, we found RcH1.1 and RcH1.3 may be under balancing selection.

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Isolation and analysis of linker histones across cellular compartments.

Publications

Isolation and analysis of linker histones across cellular compartments.

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