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 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.

Epigenetics and autism spectrum disorder: A report of an autism case with mutation in H1 linker histone HIST1H1E and literature review

“…They have a tripartite structure consisting of a short N‐terminus enriched in basic amino acids, a central conserved globular H15 domain important for DNA binding, and a long C‐terminal tail enriched in lysine, serine, and proline. H1 linker histone proteins are believed to contribute to the organization and stabilization of chromosomal DNA, in addition to the folding of nucleosome filaments into higher‐order structures and modulation of gene transcription (Happel & Doenecke, ; Harshman et al, ; Harvey & Downs, ; Trollope, Sapojnikova, Thorne, Crane‐Robinson, & Myers, ; Xiao, Freedman, Miller, Heald, & Marko, ). Mouse models knocking out expression of various linker histones, including specific H1 subtypes, confirm these proteins are involved in regulating specific genes and specific processes (Sancho, Diani, Beato, & Jordan, ).…”

Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence

“…Extracted histones were dissolved in 350 l mobile phase A (95% H 2 O, 5% CH 3 CN, 0.2% TFA) and loaded onto a 4.6 ϫ 250 mm, 5 m, 300 Å pore size C 18 column (218TP52, GRACE/Vydac, Columbia, MD) as reported elsewhere (57)(58)(59). Mobile phase B consisted of 5% H 2 O, 95% CH 3 CN, and 0.188% TFA, increasing linearly from 5% to 30% in 5 min, and then increasing 0.3% B/min to 60% at 105 min.…”

Modulation of chromatin function through linker histone H1 variants

“…the mitosis (Harshman et al, 2013a). The phosphorylation status of histone H1 subtypes in human lymphoblastic T-cells (Sarg et al, 2006) was shown to be different during interphase and mitosis.…”

“…Usually, histone H1 modifications have functional implications. Phosphorylation, customarily linked with chromatin condensing capacity and transcriptional activity (Strahl and Allis, ), was also recognized to be uniquely coupled to the progression of cell cycle (Gurley et al., ), reaching its maximum level at the mitosis (Harshman et al., ). The phosphorylation status of histone H1 subtypes in human lymphoblastic T‐cells (Sarg et al., ) was shown to be different during interphase and mitosis.…”

“…In addition to the histone H1 tail modifications, the modification sites located within the globular domain were also mapped (Wiśniewski et al, 2007;Harshman et al, 2013a). Moreover, methylated lysine (K26) and adjacent phosphorylated serine (Ser27) residues were detected in a histone H1.4 peptide from HeLa cells (Garcia et al, 2004).…”