Activity banding of human chromosomes as shown by histone acetylation

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromsomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromsomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a “snap-shot” of the distribution of gene activity on chromosomes at the time of cell harvest. Edited by: P.B. Moens     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.     

Butyrate-induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid-extracted histones of control and butyrate-treated cells were analyzed by two-dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate-treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.     

Histone variants and histone modifications in chromatin fractions from heterochromatin-rich Peromyscus cells

In order to investigate the relationship between condensed heterochromatin and histone modification by acetylation, phosphorylation and amino acid variation, chromatin from cultured Peromyscus eremicus cells, containing 35% constitutive heterochromatin, was fractionated into heterochromatin-enriched and heterochromatin-depleted fractions. The constitutive heterochromatin content of these fractions was determined from satellite DNA content. The distribution of phosphorylated and acetylated histones and amino acid variants of histone H2A in these chromatin fractions was examined by gel electrophoresis. Fractionation of histones demonstrated that endogenous histone phosphatase activity was high in chromatin fractions and could not be inhibited sufficiently to allow accurate histone phosphorylation measurements. However, sodium butyrate did inhibit deacetylation activity in the fractions, allowing histone acetylation measurements to be made. It was found that the constitutive heterochromatin content of these fractions was proportional to both their unacetylated H4 content and their more-hydrophobic H2A content. These observations support, by direct measurement, earlier experiments (Exp cell res 111 (1978) 373; 125 (1980) 377; 132 (1981) 201) suggesting that constitutive heterochromatin is enriched in unacetylated arginine-rich histones, and in the more hydrophobic variant of histone H2A.     

C- and Q-chromatin of the experimentally condensed human interphase chromosomes]

Condensed interphase chromosomes of the cultured human lymphocytes obtained by the fusion of interphase and metaphase cells were studied using C- and Q-bands techniques. The appearance and localization of the constitutive heterochromatin blocks on condensed chromosomes at G1-period were the same as on the metaphase ones. These characters were used for a group and individual identification of some chromosomes condensed at G1-period and for a study of the association of the constitutive heterochromatin blocks in the interphase nuclei. The fluorescent analysis of the chromosomes condensed at G1-period detected some bright fluorescent blocks of the constitutive heterochromatin.     

Length of human C-bands in relation to the degree of chromosome condensation

Summary The C-band length of human chromosome 1 in prophase and prematurely condensed interphase chromosomes is relatively shorter than in metaphase chromosomes. However, even in chromosomes with the same degree of contraction the absolute length of the C-band varies considerably. This allocyclic behaviour of human constitutive heterochromatin has to be kept in mind if C-bands of different individuals are compared.Sponsored by the Deutsche Forschungsgemeinschaft (Sp 144)     

DNA associated with hyperacetylated histone is preferentially digested by DNase I.

Butyrate-treated cells give rise to massive hyperacetylation of histones and have been used to test the idea that regions of DNA in association with hyperacetylated histones are preferentially solubilized upon digestion with DNase I. Such hyperacetylated histones can be derived from both pre-existing histones or from histone newly synthesized in the presence of butyrate which leads to extreme modification. The DNA in association with both types of hypermodified histone is equally and selectively digested.     

Conservation of the acetylation pattern of histones and the transcriptional activity in Ehrlich ascites tumor cells by sodium butyrate

Histone deacetylases of Ehrlich ascites tumor cells are active at low temperatures (0-4 degrees C). The so-called hyperacetylated state of histones is the physiological state of histones in intact Ehrlich ascites tumor cells which is conserved by the continuous presence of 10 mM sodium butyrate during the preparation of nuclei and histones. Isolation of histones in the absence of butyrate causes an artificial decrease in histone acetylation. This artificial loss of histone acetylation produces a decrease of the elongation reaction in the RNA synthesis. The initiation of RNA synthesis is not affected.     

Histone H2A subfractions and their phosphorylation in cultured Peromyscus cells

Patterns of histone phosphorylation and histone H2A subfractionation have been compared in cultured cell lines from two species of deer mice, Peromyscus eremicus and Peromyscus boylii, which differ considerably in their content of heterochromatin but which contain essentially the same euchromatin content. DNA measurements by flow microfluorometry indicated that P. eremicus cells contained 34.2% more DNA than P. boylii cells, and C-band chromosome analysis indicated that the extra DNA in P. eremicus was present as constitutive heterochromatin. Subfraction of histone H2A by acid-urea polyacrylamide preparative gel electrophoresis in the presence of non-ionic detergent showed that each cell line contained two H2A subfractions. Incorporation of ³²PO₄ into these histones indicated that the steady state phosphorylation of the two H2A subfractions was not the same, the more hydrophobic H2A being greater than two times more phosphorylated than the less hydrophobic H2A in both cell lines. A comparison of the two cell lines indicated that the cell line with 34.2% greater constitutive heterochromatin contained a similar excess (29%) in its ratio of the more highly phosphorylated, more hydrophobic H2A subfraction to the less hydrophobic H2A subfraction. It is suggested that this enrichment of the more highly phosphorylated, more hydrophobic H2A subfraction may be related to the amount of constitutive heterochromatin present in the genome.     

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.     

Histone hyperacetylation has little effect on the higher order folding of chromatin

HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation. Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation. Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid.     

Histone deacetylation in nuclei isolated from hepatoma tissue culture cells. Inhibition by sodium butyrate.

Nuclei from hepatoma tissue culture (HTC) cells were isolated by standard methods and incubated in media commonly used for nuclease digestions (DNAase I and micrococcal nuclease) and for in vitro RNA synthesis. During the incubation, histones can be deacetylated from both control cells and cells treated with 6 mM sodium butyrate to enhance the levels of histone acetylation. Deacetylation of histone is much more apparent in nuclei isolated from sodium butyrate-treated cells. Inclusion of 6 mM sodium butyrate in the incubation medium effectively inhibits the endogenous deacetylase activity acting on histones H3 and H4, whereas sodium acetate at the same concentration has very little inhibitory effect.     

Histone deacetylation in nuclei isolated from hepatoma tissue culture cells Inhibition by sodium butyrate

Nuclei from hepatoma tissue culture (HTC) cells were isolated by standard methods and incubated in media commonly used for nuclease digestions (DNAase I and micrococcal nuclease) and for in vitro RNA synthesis. During the incubation, histones can be deacetylated from both control cells and cells treated with 6 mM sodium butyrate to enhance the levels of histone acetylation. Deacetylation of histone is much more apparent in nuclei isolated from sodium butyrate-treated cells. Inclusion of 6 mM sodium butyrate in the incubation medium effectively inhibits the endogenous deacetylase activity acting on histones H3 and H4, whereas sodium acetate at the same concentration has very little inhibitory effect.     

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.     

Heterochromatin and histone phosphorylation

Histone phosphorylation and nuclear structure have been compared in cultured cell lines of two related species of deer mice, Peromyscus crinitus and Peromyscus eremicus, which differ greatly in their heterochromatin contents but which contain essentially the same euchromatin content. Flow microfluorometry measurements indicated that P. eremicus contained 36% more DNA than did P. crinitus, and C-band chromosome staining indicated that the extra DNA of P. eremicus existed as constitutive heterochromatin. Two striking differences in interphase nuclear structure were observed by electron microscopy. Peromyscus crinitus nuclei contained small clumps of heterochromatin and a loose, amorphous nucleolus, while P. eremicus nuclei contained large, dense clumps of heterochromatin and a densely structured, well defined, nucleolonema form of nucleolus. Incorporation of ³²PO₄ into histones indicated that the steady-state phosphorylation of H1 was identical in P. crinitus and P. eremicus cells. In contrast, the phosphorylation rate of H2a was 58% greater in the highly heterochromatic chromatin of P. eremicus cells than in the lesser heterochromatic chromatin of P. crinitus cells, suggesting an involvement of H2a phosphorylation in heterochromatin structure. It is suggested that the three histone phosphorylations related to cell growth (H1, H2a, and H3) may be associated with different levels of chromatin organization: H1 interphase phosphorylation with some submicroscopic (molecular) level of organization, H2a phosphorylation with a higher level of chromatin organization found in heterochromatin, and H3 and H1 superphosphorylation with the highest level of chromatin organization observed in condensed chromosomes.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.     

Variability and familial transmission of constitutive heterochromatin of human chromosomes evaluated by the method of linear measurement

Summary Using the method of linear measurement, the lengths of constitutive heterochromatin of chromosomes 1, 9, 16, and Y were determined in 125 unrelated individuals, and in 30 members of ten families. The method used eliminates the variations in the C-band length due to different degrees of contraction of chromosomes in different mitoses, and enables the size of heterochromatin blocks to be expressed. It was found that the distribution of C-band lengths in the group of 125 individuals was normal, i.e., Gaussian, for all four classes of chromosomes measured. On the basis of length distribution and by computing the P₁, P₁₀, P₉₀ and P₉₉ percentiles, the actual numerical limits could be proposed for the five-step evaluation of heterochromatin length according to the Paris Conference (1971), Supplement (1975), for chromosomes 1, 9, 16, and in a preliminary way also for Y. When applying the proposed limits to data obtained in the present study, 165 C-band variants could be identified among the 125 individuals.In ten families, C-block lengths of the chromosomes transmitted from parents to progeny could be determined in 63 cases. The mean difference in C-band length of transmitted chromosomes, as measured in parents and in children, was 0.46×10^-7 m. An analysis was carried out to detect the factors upon which the magnitude of this difference depends, and to define what differences are attributable to methodological errors. The results revealed that the difference rises slightly with the increasing length of the measured C block. Three degrees, defined by concrete ranges of difference in C-block length, were proposed for expressing the probability that the compared chromosomes had been transmitted.The study further attests to the effectiveness of the method of constitutive heterochromatin measurement for paternity testing. In our set of ten families, the comparison of C-band lengths of chromosomes 1, 9, 16, and Y led to rejection of paternity in 64% of unrelated individuals; excluding the Y chromosome, the percentage decreased to 61. As many as 47% of the individuals were rejected by a difference higher than two units (i.e., transmission of the compared chromosome highly improbable).     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Histone H1 in nuclei of butyrate-treated murine lymphosarcoma cells has increased affinity for heparin

Nuclei from butyrate-treated murine lymphosarcoma cells were incubated with different amounts of the polyanion heparin, which is known to interact predominantly with chromatin-associated histones. Unlike isolated histone H1, histone H1 in the nuclei of butyrate-treated cells was found to display an enhanced affinity for the binding to heparin as compared to histone H1 from control cells. Dephosphorylation of histone H1 as a result of butyrate treatment of the cells is discussed as a possible factor involved in the observed higher affinity of the protein for heparin.