Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment.
Staining was visualized with FITC-conjugated donkey anti–rabbit antibody (1:200; Jackson ImmunoResearch Laboratories, Inc.) and Cy3-conjugated donkey anti–mouse antibody (1:800; Jackson ImmunoResearch Laboratories, Inc.). H1.2 contains four Cdk substrate motifs, but MS/MS analysis of the 1p-H1.2 peak localized phosphorylation exclusively to S173 in both allelic variants.
Our findings also suggest that interphase H1 kinases preferentially phosphorylate Ser-containing Cdk substrate motifs, which is consistent with evidence that three such sites in H1.5 are phosphorylated during interphase in human cells (To investigate the roles of specific H1 phosphorylations, we generated antisera against phosphopeptides containing the pS173-H1.2 and pS187-H1.4 I sites and the pS27-H1.4 and pT154-H1.4 M sites (Fig. Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 FRAP analyses of cells expressing H1-GFP fusions have revealed that H1 variants bind chromatin dynamically in vivo and that both the globular domain and CTD contribute to chromatin binding (CTD interactions with linker DNA are important for higher order folding of chromatin (Human somatic cells express six H1 variants with distinct chromatin-binding dynamics that possess CTDs differing in length, net charge, number, and relative positions of S/TPXK/R motifs (We used top-down mass spectrometry (MS [TDMS]) to analyze H1 phosphorylation because this approach facilitates characterization of multisite histone modification (To enhance phosphorylation site identification, we used hydrophobic interaction chromatography (HIC) to enrich phosphorylated forms of H1 before TDMS. Ann M. Nardulli The specificity of these antisera was validated using Western blotting (Fig. This has been connected to increasing phosphorylation of H1 histones through the cell cycle. Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Notably, monophosphorylation at H1.4-S172 was not detected, implying that interphase H1.4 phosphorylation/dephosphorylation occurs hierarchically or that other mechanisms prevent the accumulation of detectable H1.4-S172 monophosphorylation.
We investigated this question in the SV40 in vitro replication system, by using salt-treated SV40 minichromosomes, reconstituted with G0-, S- and M-phase histone H1, as template for replication in … An extra-nucleosomal histone, the linker histone H1, is also known to be phosphorylated on multiple residues. R. Louis Schiltz Search for other works by this author on: Phosphorylation of H1 increases during the transition from G 1 to S phase, reaching a limited maximum during S phase . Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Recombinant human H1.4 was complexed with yeast tRNA before rabbit immunization as described previously (HeLa cells grown on glass coverslips were fixed with 4% (wt/vol) paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and immunostained with primary antisera using standard procedures. Kinases that mediate interphase H1 phosphorylation in vivo have not been directly identified, but several lines of evidence implicate Cdk2 (HeLa S3 cells were grown in suspension in Joklik’s modified minimal essential medium supplemented with 10% newborn calf serum (NCS) and synchronized using the double-thymidine block procedure as described previously (Crude H1 was prepared by 5% perchloric acid fractionation of crude histones (Reverse phase HPLC used a column (4.6 mm ID × 250 mm; Vydac C18) with a multistep linear gradient from buffer A (0.1% vol/vol TFA in 5% vol/vol CHMS data were acquired on a custom 8.5-T quadrupole Fourier transform ion cyclotron resonance mass spectrometer with an electrospray ionization source operated in positive-ion mode as described previously (Phosphorylated and nonphosphorylated peptides were custom synthesized. Kelleher (GM 067193) and A.M. Nardulli (R01 DK53884). S2 shows the specificity of antisera for individual H1 phosphorylation sites, the antisera to total H1.4 in immunoblots, and the phosphorylation sites identified in this study relative to an alignment of the human H1.1–H1.5 protein sequences. Search for other works by this author on: During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G1. The reason for these multiple isoforms remains unclear, but both their evolutionary conservation from sea urchin to humans as well as significant differences in their amino acid sequences suggest that they are not functionally equivalent.Like other histones, the histone H1 family is extensively post-translationally modified (PTMs).