H1 Depletion Research Articles

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.

Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G 1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901–1910). During the time that synchronized G 1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.

Fractionation of chromatin fragments on columns of Biogel A50-m at different salt concentrations

Nuclease fragmented chromatin was chromatographed on Biogel at various NaCl concentrations. The yield of eluted chromatin, and its H1/core histone ratio was minimal at 0.18 M NaCl where the ratio of H1 subtypes H1c/H1 ab was maximal. Therefore, the eluted material was aggregation-resistant chromatin while aggregatable chromatin remained on the columns. Previous results were interpreted as Hl depletion of chromatin by ion-exchange properties of Biogel, but the primary phenomenon is now seen as a separation of classes of chromatin that differ in sensitivity to salt-induced aggregation. At very low salt concentrations, Biogel chromatography can be used without concern for H1 depletion.

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin. These morphological features are accompanied by depletion of lysine-rich histones H1 and H1(0) and of histone-like protein A5, due to PS liposomes. Functionally, PS vesicles induce enhancement both of total RNA and specific mRNA synthesis, as analysed by in vitro transcription of beta globin gene. On the contrary, PC vesicles do not seem to affect significantly total RNA and globin mRNA synthesis. These observations fit well with previous data obtained in other experimental systems, and support both the use of these molecules as probes for chromatin structure and function and their possible involvement in transcriptional events.

Histone H1 in G1 arrested, senescent, and Werner syndrome fibroblasts.

Histone H1 content and synthesis were examined in normal, Werner-syndrome, and transformed fibroblasts. Analysis of 3H-lysine incorporation indicated that senescent cells, but not G1-arrested young cells, had a lower ratio of molar synthesis of H1 histone to nucleosome histones than did growing young cells or gamma-ray-transformed cells. Furthermore, a biochemical study of histone H1 content plotted as a function of DNA synthesis activity and an immunocytological study using antiserum against histone H1 revealed that senescent cells had a lower histone H1 content than did young cultures at all stages of cell proliferation. Werner syndrome skin fibroblasts at early passage, however, had amounts of histone H1 comparable to those of age-matched normal control fibroblasts. We conclude that a decline, with increasing passage number, in content and synthesis of H1 histone relative to nucleosomal histones (Mitsui et al., 1980) was not simply due to passage-related accumulation of G1-arrested cells, but actually reflected age specific changes of cultured human fibroblasts. The depletion of histone H1 in the chromatin of senescent cells is a possible cause of DNA strand breakage or relaxation of gene repression.

The Structural Organization of Oligonucleosomes

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin. The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r = 0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.

The effect of low ionic strength on the circular dichroic spectrum of chromatin and nucleosomal subunits

Circular dichroism has been used to measure the conformation changes in the DNA of chromatin and chromatin subunits as a function of ionic strength. Transfer of chromatin from 0.15 m to 0.25 m m salt led to an enhancement of the circular dichroic bands at 275 and 285 nm. Removal of histone H1 did not appreciably affect the circular dichroic spectrum when measured in 0.15 m salt, but in 0.25 m m salt H1 depletion led to a marked increase in the ellipticity. Conformation changes due to low ionic strength were also observed with a 145- and a 172-bp chromatin subunit. A linear combination of the ellipticities of the DNA of the two domains in chromatin, namely core and linker, was successful for measurements at 0.15 m salt, but large unexplained discrepancies appeared with the data from measurements in 0.25 m m salt.

Histone H1 depletion with human cellular aging: Biochemical and cytochemical evidences.

Biochemical and immunocytochemical examinations of histone H1 in G1 arrested and senescent human fibroblasts were performed to pursuit the mechanisms of human cell aging in vitro. Examinations of histone synthesis in serum depleted or contact inhibited cell cultures indicated that the ratio of histone synthesis over DNA synthesis and molar synthesis of histone H1 to nucleosomal histones did not decline with accumulation of resting cell populations, while senescent cells had a lower ratio of histone H1 synthesis than young cells. Histone H1 contents plotted as a function of DNA synthesis activity revealed that senescent cultures had a lower content of histone H1 than young cultures at any stage of cell proliferation.Immunocytological study using antiserum against histone H1 on cocultivated young and senescent cells showed that immunofluorescence intensity was very low in senescent nuclei in comparison with young cell nuclei. Thus, we conclude that a decline in content and synthesis of histone H1 relative to nucleosomal histones with passage number was not simply due to the passage-related accumulation of resting cells, but actually reflects age-specific changes in cycling cells or whole cell populations in the senescent cultures. The depletion of histone H1 in chromatin was discussed as a possible cause of DNA strand break and relaxation of gene repression.

Activation of the major Drosophila heat-shock genes in vitro

Inactive Drosophila heat-shock genes of isolated diploid nuclei can be induced to a transcriptionally active state by exposure to cytoplasmic extracts from heat-shocked Drosophila cells. No effect was observed on histone gene transcription, and extracts from non-heat-shocked cells were ineffective. The factor in the cytoplasmic extract has been partially purified and characterized. It is protease-sensitive and heat-labile. A striking change accompanies in vitro activation that permits transcription by E. coli RNA polymerase of the chromatin 5′-distal to the structural genes at the 87A and 87C heat-shock gene loci; we have previously observed a similar change after in vivo heat-shock induction. That this change occurred in the absence of endogenous RNA polymerase II activity suggests that these changes may reflect the earliest event in gene activation. Inasmuch as activation also took place after histone H1 depletion, this histone does not appear to be essential for this step of gene activation.

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in ( i) nuclei depleted of H1 by acid extraction; ( ii) nuclei treated at pH 3.2 (without removal of H1), and ( iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.

Micrococcal nuclease cleavage of chromatin displays nonrandom properties.

A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed. Some possible mechanisms to account for this property are discussed.