H1 Content Research Articles

Quantification of Histone H1 Subtypes Using Targeted Proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response.

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

TADs enriched in histone H1.2 strongly overlap with the B compartment, inaccessible chromatin, and AT-rich Giemsa bands.

Giemsa staining of metaphase chromosomes results in a characteristic banding useful for identification of chromosomes and its alterations. We have investigated insilico whether Giemsa bands (G bands) correlate with epigenetic and topological features of the interphase genome. Staining of G-positive bands decreases with GC content; nonetheless, G-negative bands are GC heterogeneous. High GC bands are enriched in active histone marks, RNA polymerase II, and SINEs and associate with gene richness, gene expression, and early replication. Low GC bands are enriched in repressive marks, lamina-associated domains, and LINEs. Histone H1 variants distribute heterogeneously among G bands: H1X is enriched at high GC bands and H1.2 is abundant at low GC, compacted bands. According to epigenetic features and H1 content, G bands can be organized in clusters useful to compartmentalize the genome. Indeed, we have obtained Hi-C chromosome interaction maps and compared topologically associating domains (TADs) and A/B compartments to G banding. TADs with high H1.2/H1X ratio strongly overlap with B compartment, late replicating, and inaccessible chromatin and low GC bands. We propose that GC content is a strong driver of chromatin compaction and 3D genome organization, that Giemsa staining recapitulates this organization denoted by high-throughput techniques, and that H1 variants distribute at distinct chromatin domains. DATABASES: Hi-C data on T47D breast cancer cells have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE147627.

Irrigation method and harvest time affect storage quality of two early-season, tablestock potato (Solanum tuberosum L.) cultivars

Storage quality was evaluated for two tablestock potato (Solanum tuberosum L.) cultivars grown in a sub-tropical climate under three irrigation methods and harvested over a three-week period. Early-maturing ‘Fabula’ and ‘Red LaSoda’ potatoes were irrigated with either seepage (SE), surface drip (SD), or sub-surface drip (Sub-SD; tape buried 15cm) irrigation, during two spring seasons. Inherent potato seed dormancy in ‘Fabula’ resulted in delayed and non-uniform plant establishment. At horticultural maturity, vines were killed using a chemical desiccant to promote tuber periderm maturation. Tubers were harvested 1–3 weeks after vine kill (H1, H2, H3), and tuber quality was evaluated weekly during 14 days storage at 10°C and 80–85% relative humidity (RH). In ‘Fabula’, H1 tubers, with more immature periderms, had the highest weight loss (5.3%) and were significantly less firm after 14 days of storage. Average cumulative weight loss for later harvests was 1.4% and tubers remained firm throughout storage. Pulp (cortex, xylem, perimedullary and pith tissue) ascorbic acid content of H1 ‘Fabula’ tubers decreased during storage; however, it was higher in SD and Sub-SD (17.1mg 100g−1) than in SE tubers (11.8mg 100g−1) after 14 days. There were no significant differences in whole tuber or peel ascorbic acid content during storage; average was 20.1mg 100g−1 and 7.2mg 100g−1, respectively. Although dry matter content in the peel decreased with increased delay to harvest time for all irrigation methods, it remained constant throughout storage. Average dry matter content, for all harvest times, during storage was 16.7%, 8.8% and 14.8% for whole tuber, peel and pulp tissue, respectively. ‘Red LaSoda’ plants tend to grow faster than ‘Fabula’ and, therefore were compositionally more mature at harvest; no significant differences in dry matter or ascorbic acid content were observed before or during storage. However, similar to ‘Fabula’ tubers at H1, firmness decreased by 7 days in storage for all irrigation methods, possibly due to excessive fresh weight loss. Harvest and storage quality of ‘Fabula’ and ‘Red LaSoda’ grown under either SD or Sub-SD was comparable to SE at H2 and H3, with the potential to significantly reduce irrigation water usage during production.

Genome Distribution of Replication-independent Histone H1 Variants Shows H1.0 Associated with Nucleolar Domains and H1X Associated with RNA Polymerase II-enriched Regions

Unlike core histones, the linker histone H1 family is more evolutionarily diverse, and many organisms have multiple H1 variants or subtypes. In mammals, the H1 family includes seven somatic H1 variants; H1.1 to H1.5 are expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Using ChIP-sequencing data and cell fractionation, we have compared the genomic distribution of H1.0 and H1X in human breast cancer cells, in which we previously observed differential distribution of H1.2 compared with the other subtypes. We have found H1.0 to be enriched at nucleolus-associated DNA repeats and chromatin domains, whereas H1X is associated with coding regions, RNA polymerase II-enriched regions, and hypomethylated CpG islands. Further, H1X accumulates within constitutive or included exons and retained introns and toward the 3' end of expressed genes. Inducible H1X knockdown does not affect cell proliferation but dysregulates a subset of genes related to cell movement and transport. In H1X-depleted cells, the promoters of up-regulated genes are not occupied specifically by this variant, have a lower than average H1 content, and, unexpectedly, do not form an H1 valley upon induction. We conclude that H1 variants are not distributed evenly across the genome and may participate with some specificity in chromatin domain organization or gene regulation.

Revisiting axial-vector meson mixing

Various phenomenological studies indicate that the mixing angle θK1 of K1A and K1B, the strange partners of the axial-vector mesons a1(1260) and b1(1235), respectively, lies in the vicinity of 35° or 55°, but whether this angle is larger or smaller than 45° still remains controversial. When the f1(1285)–f1(1420) mixing angle θP13 and the h1(1170)–h1(1380) mixing angle θP11 are determined from the mass relations, they depend on the masses of K1A and K1B, which in turn depend on the mixing angle θK1. We show that the approximate decoupling of the light qq¯ state from the heavier ss¯ state, which is empirically valid for vector, tensor and 3−− mesons, when applied to isoscalar axial-vector mesons, will enable us to discriminate different solutions of θP13 and θP11 and pick up θK1∼35°. Indeed, for θK1∼55°, the predicted θP11 disagrees sharply with the recent lattice calculation and the implied large ss¯ content of h1(1170) and qq¯ component of h1(1380) cannot explain the observation of their strong decays. We conclude that θK1 is smaller than 45°.

Interaction between non-histone chromatin protein HMGB1 and linker histone H1

The fundamental possibility of interaction between non-histone chromatin protein HMGB1 and linker histone H1 was studied in the solutions with different ionic strength by intrinsic UV-fluorescence, far and near-UV CD and spectrophotometry. The obtained data allow us to assume that the increase of histone H1 content in the HMGB1 solutions in a low ionic strength is accompanied by the destruction of HMGB1 associates. The interaction between proteins of HMGB1 and H1 causes the increase in the number of ordered regions in the protein molecules and the minor changes in their tertiary structure.

DNA-unstable decaploid mouse H1 (ES) cells established from DNA-stable pentaploid H1 (ES) cells polyploidized using demecolcine

DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA-unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA-stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability. 5H1 cells were polyploidized using demecolcine (DC) and 10H1 cells were obtained by one-cell cloning. Number of chromosomes of 10H1 cells was 180 and durations of their G(1), S, and G(2)/M phases were 3, 7 and 6 h respectively. Volume of 10H1 cells was double that of 5H1 cells and morphology of 10H1 cells was flagstone-like in shape. 10H1 cells exhibited alkaline phosphatase activity and their DNA content decayed in 91 days of culture. 10H1 cells injected into mouse abdomen formed solid tumours that contained several kinds of differentiated cells with lower DNA content, suggesting that 10H1 cells were pluripotent and DNA-unstable. Loss of DNA stability was explained using a hypothesis concerning DNA structure of polyploid cells as DNA reconstructed through ploidy doubling was arranged in mirror symmetry in a new configuration. In the pentaploid-decaploid transition of H1 cells, cell cycle parameters and pluripotency were retained, but morphology and DNA stability were altered.

Interaction between linker histone H1 and non-histone chromatin protein HMGB1

The possibility of interaction between linker histone H1 and non-histone chromatin protein HMGB1 was studied by intrinsic UV-fluorescence, far and near-UV CD and light scattering. The obtained data allow us to assume that the increase of histone H1 content in the HMGB1 solutions in a low ionic strength is accompanied by the destruction of HMGB1 associates. The interaction between proteins causes the increase of ordered regions in the protein molecules and the minor changes in their tertiary structure.

Interaction between linker histone H1 and non-histone chromatin protein HMGB1

The possibility of interaction between linker histone H1 and non-histone chromatin protein HMGB1 was studied by intrinsic UV-fluorescence, far and near-UV CD and light scattering. The obtained data allow us to assume that the increase of histone H1 content in the HMGB1 solutions in a low ionic strength is accompanied by the destruction of HMGB1 associates. The interaction between proteins causes the increase of ordered regions in the protein molecules and the minor changes in their tertiary structure.

Evidence for Insertional RNA Editing in Humans

Large-scale analysis directly at the protein level holds the promise of uncovering features not apparent or present at the gene level [1-3]. Although mass spectrometry (MS)-based proteomics can now identify and quantify thousands of cellular proteins in large-scale proteomics experiments, much of the peptide information contained in these experiments remains unassigned [4]. Here, we use such information to discover a previously unreported mechanism creating altered protein forms. Linker histones H1 and high-mobility group (HMG) proteins are abundant nuclear proteins that regulate gene expression through modulation of chromatin structure [5-8]. In the high-resolution MS analysis of histone H1 and HMG protein fractions isolated from human cells, we discovered peptides that mapped upstream of the known translation start sites of these genes. No alternative upstream start site exists in the genome, but analysis of Expressed Sequence Tag (EST) databases revealed that these N-terminally extended (ET) proteins are due to in-frame translation of the 5' untranslated region (5'UTR) sequences of the transcripts. The new translation start sites are created by a single uridine insertion between AG, reflecting a previously unreported RNA-editing mechanism. To our knowledge, this is the first report of RNA-insertion editing in humans and may be an example of the type of discoveries possible with modern proteomics methods.

Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length

Despite a great deal of attention over many years, the structural and functional roles of the linker histone H1 remain enigmatic. The earlier concepts of H1 as a general transcriptional inhibitor have had to be reconsidered in the light of experiments demonstrating a minor effect of H1 deletion in unicellular organisms. More recent work analysing the results of depleting H1 in mammals through genetic knockouts of selected H1 subtypes in the mouse has shown that cells and tissues can tolerate a surprisingly low H1 content. One common feature of H1-depleted nuclei is a reduction in nucleosome repeat length (NRL). Moreover, there is a robust linear relationship between H1 stoichiometry and NRL, suggesting an inherent homeostatic mechanism that maintains intranuclear electrostatic balance. It is also clear that the 1 H1 per nucleosome paradigm for higher eukaryotes is the exception rather than the rule. This, together with the high mobility of H1 within the nucleus, prompts a reappraisal of the role of linker histone as an obligatory chromatin architectural protein.

Changes in Composition of Acid Soluble Proteins and DNA in Chromatin of Rat Liver and Brain Bound and Not Bound to Nuclear Envelope as a Function of Age and under the Influence of Antioxidant Ionol

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.

Histone H2A Ubiquitination Does Not Preclude Histone H1 Binding, but It Facilitates Its Association with the Nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Content of the Hmg-17 Chromosomal Protein in Porcine Tissues

HMG-17 proteins are nucleosomal proteins, implicated in control of chromatin structure and transcription, in a way that has not yet been fully understood. In this report, quantification of HMG-17 in porcine tissues was performed, by ELISA, using previously produced and characterized specific rabbit anti-HMG-17 antibodies. Our results showed high levels of HMG-17 compared to the DNA and H1 content of the tissues (on the contrary to previous reports), and more specifically that there were 1.7 molecules of HMG-17 per molecule of histone H1.

H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.

Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by mid-gestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes.

A mechanistic study of the histone H1-DNA complex dissociation by sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on H1-DNA complex is studied by different methods. The histone H1 fluorescence emission increases on SDS addition. It is may be due to the transfer of the only tyrosine residue of H1 to a more hydrophobic environment. Circular dichroism (CD) shows an increase of 17.1% in α-helix content of H1 in the presence of SDS. The CD spectrum of DNA in the presence of SDS also indicates the transfer of DNA from the B-form to the A-form both in the absence and presence of H1. The isothermal titration calorimetric investigation indicates that H1–SDS and DNA–SDS interactions are both exothermic. Spectrophotometric study of the percent of precipitation of DNA by H1 has shown that the complex formation is highly cooperative and it is completely dissociates by SDS >0.5 mM. The results signify two important reasons for H1-DNA complex dissociation: (1) the addition of SDS reduces the ability of H1 to neutralize the negative charges of DNA, and (2) concurrently the hydrophobic chains of SDS induce the DNA conformational changes. Furthermore, the precipitation curve in the presence of lower concentrations of SDS (in the range of 0.25–0.5 mM) shows two domains. The calorimetric titration curve of DNA by H1 or the folded H1 (by 0.4 mM SDS) also reveal two domains. The results indicate that the C-terminal domain of intact H1 possibly is the first domain that is dissociated from DNA and then the separation of the globular domain occurs in the presence of SDS.

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.

Study of a Slice at +9° to +15° of Declination. I. The Neutral Hydrogen Content of Galaxies in Loose Groups

We examine the H1 content of spiral galaxies in groups by using a catalog of loose groups of galaxies identified in a magnitude limited sample m < 15.7 spanning the range 8 h to 18 h in right ascension and +9 to +15 in declination. The redshift completeness of the galaxy sample is ~95%. No significant effect of H1 depletion is found, although there may be a hint that the earliest type spirals are slightly deficient.

Condensation of Rat Telomere-specific Nucleosomal Arrays Containing Unusually Short DNA Repeats and Histone H1

Vertebrate telomeres contain arrays of nucleosomes with unusually short and regular repeat lengths (Makarov, V. L., Lejnine, S., Bedoyan, J., and Langmore, J. P.(1993) Cell 73, 775-787; Lejnine, S., Makarov, V., and Langmore, J. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2393-2397). In order to better define the specific structural features of telomere chromatin, we examined the condensation and H1 content of telomere nucleoproteins from rat liver. Velocity sedimentation analysis shows that telomeric nucleosome arrays condense with increasing ionic strength and molecular weight in a manner comparable with that of bulk chromatin despite the very short repeat length. However, these condensed structures do not exhibit the approximately 100-base pair deoxyribonuclease II repeat characteristic of condensed bulk chromatin. Frictional coefficient calculations suggest that telomere-specific higher order structure is more compact than bulk chromatin. Nucleoprotein gel electrophoresis shows that telomeric dinucleosomes from soluble chromatin contain H1. Finally, direct isolation and analysis of telomere nucleoproteins from formaldehyde-cross-linked nuclei indicate the presence of core histone proteins and H1. These results are consistent with the view that a major fraction of the long telomeres of rat are organized as specialized nucleosome arrays with features similar but not identical to those of bulk chromatin.