Cytochemical properties of euchromatin and heterochromatin

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Histone modification in constitutive heterochromatin versus unexpressed euchromatin in human cells

Histone modifications are implicated in regulating chromatin condensation but it is unclear how they differ between constitutive heterochromatin and unexpressed euchromatin. Chromatin immunoprecipitation (ChIP) assays were done on various human cell populations using antibodies specific for acetylated or methylated forms of histone H3 or H4. Analysis of the immunoprecipitates was by quantitative real-time PCR or semi-quantitative PCR (SQ-PCR). Of eight tested antibodies, the one for histone H4 acetylated at lysine 4, 8, 12, or 16 was best for distinguishing constitutive heterochromatin from unexpressed euchromatin, but differences in the extent of immunoprecipitation of these two types of chromatin were only modest, although highly reproducible. With this antibody, there was an average of 2.5-fold less immunoprecipitation of three constitutive heterochromatin regions than of four unexpressed euchromatic gene regions and about 15-fold less immunoprecipitation of these heterochromatin standards than of two constitutively expressed gene standards (P <0.001). We also analyzed histone acetylation and methylation by immunocytochemistry with antibodies to H4 acetylated at lysine 8, H3 trimethylated at lysine 9, and H3 methylated at lysine 4. In addition, immunocytochemical analysis was done with an antibody to heterochromatin protein 1alpha (HP1alpha), whose preferential binding to heterochromatin has been linked to trimethylation of H3 at lysine 9. Our combined ChIP and immunocytochemical results suggest that factors other than hypoacetylation of the N-terminal tails of H4 and hypermethylation of H3 at lysine 9 can play an important role in determining whether a chromatin sequence in mammalian cells is constitutively heterochromatic.     

Replication of euchromatin and heterochromatin in a mealybug

Asynchronous DNA replication of euchromatic (E) and heterochromatic (H) chromosomes and heterochromatic B chromosomes (B) were studied in the mealybug, Pseudococcus obscurus Essig (Homoptera: Coccoidea). The study was carried out on mycetocytes of adult females and on spermatocytes of mid-second instar males by employing tritiated thymidine labeling and autoradiography. In the mycetocytes the incorporation of the labeled thymidine began and ended later in the B's than in the E chromosomes. The S period was found to be about 21 hours. The DNA replication of the E chromosomes occupied about 86% of the S period and that of the B's 33%; during 18% of the mid-S period the replication of the two types of chromosomes overlapped. In the meiotic S period of the spermatocytes, the DNA of the E chromosomes started to replicate earlier than that of the H chromosomes and the B's, but the replication of the E chromosomes, the H chromosomes, and the B's overlapped. The H chromosomes completed their replication much later than the E chromosomes and slightly later than the B's.Supported by grants GB 1585 and GB 6745 to Dr. Uzi Nur from the National Science Foundation, Washington, D. C.Part of a thesis submitted to the University of Rochester in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Interaction systems between heterochromatin and euchromatin inDrosophila melanogaster

The constitutive heterochromatin is still one of the major unsolved problems in genetics. InDrosophila melanogaster three genetic systems involving specific interactions between heterochromatic and euchromatic genetic elements are known: the Segregation Distortion, thecrystal-Stellate and theabo-ABO systems. The genetic and molecular analysis of each system will allow the identification of all the components and the elucidation of the mechanisms underlying their interactions. The results of this analysis should provide insights into the biological significance of heterochromatin and into the evolutionary forces that result in the maintainance and stability of this enigmatic genetic material.     

Feulgen-DNA absorption spectrum of euchromatin and constitutive heterochromatin.

When the heterochromatin and euchromatin of Triatoma infestans are maximally depurinated, their Feulgen-DNA spectral profiles exhibit a shoulder at about lambda = 530 nm. The shoulder is especially prominent in the curves of the heterochromatin. It is assumed that this pattern is related to richness in repetitive DNA in the heterochromatin, favoring appearance of Schiff reagent molecules di-substituted with adjacent apurinic acid aldehydes.     

Differential rates of sister chromatid exchanges between euchromatin and heterochromatin

In two rodent species, the Chinese hamster and the montane vole (Microtus montanus), the rate of sister chromatid exchange was lower in constitutive heterochromatin than in euchromatin.     

Contrasting response of euchromatin and heterochromatin to translocation in polytene chromosomes of Drosophila melanogaster

Studies on Feulgen-DNA content in the polytene chromosomes of D. melanogaster T(14)w ^m258-21 heterozygotes showed that when the euchromatic region 3D₁-E₂ is located next to the heterochromatic breakpoint it contains less DNA than in the non-translocated homologue (Hartmann-Goldstein and Cowell, 1976). In contrast to the region adjacent to the breakpoint, region 3C_1–10, which contains intercalary heterochromatin, shows more DNA in the translocated than in the non-translocated chromosome. Transposition may induce morphologically euchromatic regions containing putatively underreplicated sequences to undergo additional replication cycles. Region 2E₁-3A₄, distal to 3C₁ and at some distance from the heterochromatic breakpoint is apparently unaffected. Extended replication and reduced DNA content in regions which have undergone chromosomal rearrangement could be accounted for by varying degrees of blockage of replication in individual strands of the polytene chromosome.     

Critical electrolyte concentration of the heterochromatin and euchromatin of Triatoma infestans

The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.     

Relative DNA content of human euchromatin and heterochromatin after G, C and Giemsa 11 banding.

Human chromosomes and interphase nuclei labeled with 3H-thymidine and treated with the ASG and trypsin technique for G banding show no DNA loss. However, after G 11 and C banding significantly more DNA is removed from euchromatin than from constitutive heterochromatin.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

A comparative study of the nonhistone proteins of rat liver euchromatin and heterochromatin

Rat liver was fractionated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by controlled shearing and glycerol gradient centrifugation. The histone and nonhistone proteins associated with each fraction were compared. No qualitative differences in histone content were observed, but heterochromatin contained 1.5 times more histone protein than did euchromatin. The nonhistone proteins of each chromatin fraction were fractionated on the basis of salt solubility into loosely bound (those extracted by 0.35 m NaCl), tightly bound (those extracted by 2.0 m NaCl), and residual nonhistone proteins (those not extracted by 2.0 m NaCl). Euchromatin contained 3.7 times more loosely bound nonhistone proteins than did heterochromatin, while the latter contained twice as much residual nonhistone protein. Euchromatin was devoid of tightly bound nonhistone protein, a component of heterochromatin. Electrophoretic analysis of these nonhistone protein fractions revealed marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix

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HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.