Content of histone H1 and histone phosphorylation in relation to the higher order structures of chromatin in Drosophila

The amount of histone H1 relative to core histones has been determined in three Drosophila species (D. melanogaster, D. texana and D. virilis) in chromatin from several tissues differing in chromatin structure and genetic activity. Low levels of H1 were found in relatively undifferentiated, early embryos as well as in a line of cultured cells. In late embryos the content of H1 was highest in D. virilis which possesses larger amounts of and a partially more compacted constitutive heterochromatin than the two other species. Polytene chromatin from larval salivary glands showed increased levels of H1 compared with diploid chromatin and the degree of phosphorylation of this histone was relatively low. The degree of phosphorylation of H2A was found to be drastically reduced in polytene as compared with diploid embryonic chromatin, which parallels the extensive underreplication of constitutive heterochromatin. Also, in diploid chromatin a qualitative correlation was observed between the relative amounts of heterochromatin and the levels of H2A phosphorylation. These findings suggest a connection between H2A phosphorylation and heavy compaction of interphase chromatin.     

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.     

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D- arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.     

Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids

Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique “meiosis-mitosis shift” at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.     

Influence of linker histone H1 on chromatin remodeling.

Chromatin-remodeling complexes have been a central area of focus for research dealing with accessing cellular DNA sequestered in chromatin. Although the linker histone H1 plays a major role in promoting and maintaining higher-order chromatin structure, it has been noticeably absent from assays utilizing chromatin-remodeling enzymes. This review focuses on two ATP-dependent chromatin-remodeling complexes, Drosophila ISWI and mammalian SWI/SNF, that have been assayed using chromatin templates containing histone H1.     

The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

In this study, we show that a reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, which is a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. We further provide evidence that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, we propose a model where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.     

Influence of histone H1 on chromatin structure

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.     

The Drosophila Df31 protein interacts with histone H3 tails and promotes chromatin bridging in vitro

Df31 is a small hydrophilic protein from Drosophila melanogaster that can act as a histone chaperone in vitro. The protein is also detected as an integral component of chromatin, present at approximately the same level as histone H1. We have developed a simple assay to measure protein binding to oligonucleosomes and used it to characterise the DF31-oligonucleosome interaction. DF31 bound to chromatin in vitro at a level comparable to that observed in vivo. The DF31-chromatin interaction required the presence of core histone tails but binding was independent of the presence of H1 in the chromatin. Multiple regions of DF31 contributed to the interaction. Df31-chromatin binding still occurred on chromatin containing only H3/4, and cross-linking experiments showed that Df31 made intimate contact with H3, suggesting that this might be the primary contact site. Finally, using immobilised chromatin templates, we showed that DF31 promoted interstrand bridging between two independent oligonucleosome chains. These results provide strong evidence for a structural role of DF31 in chromatin folding and give an indication of the mechanism involved.     

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl₂ and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.     

Effects of H1 histone variant overexpression on chromatin structure

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.