Heterochromatin Fractions Research Articles

The effects of 20-hydroxyecdysone on nuclear shape, heterochromatin quantity and gray-level co-occurrence matrix texture analysis of adrenal zona fasciculata cells in an obese gerbil (Gerbillus tarabuli) model for metabolic syndrome: a correlational study.

The aim of this study was to reveal the effects of obesity and phytotherapy with 20-hydroxyecdysone (20E) on the nuclei of adrenal zona fasciculata (ZF) in the gerbil Gerbillus tarabuli by analyzing nuclear shape and gray-level co-occurrence matrix (GLCM) texture characteristics and by quantifying heterochromatin. Twelve gerbils were divided into three groups: control (C), HC and HC-20E (animals receiving a high-calorie-diet without or with a supplement of 20E, respectively). The adrenals were removed and fixed for histological and statistical analysis. Principal component analysis showed a positive correlation of area, perimeter and textural correlation in C. Nevertheless, a negative correlation was recorded for contrast and entropy. The obesity caused a disorder in nuclear texture; negative correlation was noted with heterochromatin fraction, which may be related to increased ZF activity. However, administration of 20E seems to improve the nuclear state by preserving circularity, uniformity and homogeneity of nuclei as well as the proportion of heterochromatin, which could be a sign of a downregulation of cell activity.Our results suggest that new techniques of image processing could contribute to the understanding of nuclear changes associated with obesity and its possible therapy in this gerbil model for metabolic syndrome.

2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.

Pericentromere clustering in Tradescantia section Rhoeo involves self-associations of AT- and GC-rich heterochromatin fractions, is developmentally regulated, and increases during differentiation

A spectacular but poorly recognized nuclear repatterning is the association of heterochromatic domains during interphase. Using base-specific fluorescence and extended-depth-of-focus imaging, we show that the association of heterochromatic pericentromeres composed of AT- and GC-rich chromatin occurs on a large scale in cycling meiotic and somatic cells and during development in ring- and bivalent-forming Tradescantia spathacea (section Rhoeo) varieties. The mean number of pericentromere AT-rich domains per root meristem nucleus was ca. half the expected diploid number in both varieties, suggesting chromosome pairing via (peri)centromeric regions. Indeed, regular pairing of AT-rich domains was observed. The AT- and GC-rich associations in differentiated cells contributed to a significant reduction of the mean number of the corresponding foci per nucleus in relation to root meristem. Within the first 10 mm of the root, the pericentromere attraction was in progress, as if it was an active process and involved both AT- and GC-rich associations. Complying with Rabl arrangement, the pericentromeres preferentially located on one nuclear pole, clustered into diverse configurations. Among them, a strikingly regular one with 5–7 ring-arranged pericentromeric AT-rich domains may be potentially engaged in chromosome positioning during mitosis. The fluorescent pattern of pachytene meiocytes and somatic nuclei suggests the existence of a highly prescribed ring/chain type of chromocenter architecture with side-by-side arranged pericentromeric regions. The dynamics of pericentromere associations together with their non-random location within nuclei was compared with nuclear architecture in other organisms, including the widely explored Arabidopsis model.

Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a

BackgroundIn most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a).ResultsHere, using cell type-specific DamID we mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level.ConclusionsIn various differentiated Drosophila cell types, we discovered the existence of peripheral heterochromatin, similar to that observed in mammals. Our findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in Arabidopsis thaliana.

Microscopically visible chromatin is partitioned into two major components in Arabidopsis thaliana nuclei. On one hand, chromocenters are conspicuous foci of highly condensed “heterochromatic” domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich “euchromatin” emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes Arabidopsis a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, i.e., the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.

Evolutionary dynamics of an at-rich satellite DNA and its contribution to karyotype differentiation in wild diploid Arachis species.

Satellite DNA (satDNA) is a major component of the heterochromatic regions of eukaryote genomes and usually shows a high evolutionary dynamic, even among closely related species. Section Arachis (genus Arachis) is composed of species belonging to six different genomes (A, B, D, F, G and K). The most distinguishing features among these genomes are the amount and distribution of the heterochromatin in the karyotypes. With the objective of gaining insight into the sequence composition and evolutionary dynamics of the heterochromatin fraction in Arachis, we investigated here the sequence diversity, genomic abundance, and chromosomal distribution of a satDNA family (ATR-2) among seven diploid species of section Arachis. All of the isolated sequences were AT-rich and highly conserved at both intraspecific and interspecific levels, without any species-specific polymorphism. Pairwise comparisons of isolated ATR-2 monomers revealed that most of the nucleotide sites were in the first two transitional stages of Strachan's model. However, the abundance of ATR-2 was significantly different among genomes according to the 'library hypothesis'. Fluorescent in situ hybridization revealed that ATR-2 is a main component of the DAPI+ centromeric heterochromatin of the A, F, and K genomes. Thus, the evolution of the different heterochromatin patterns observed in Arachis genomes can be explained, at least in part, by the differential representation of ATR-2 among the different species or even among the chromosomes of the same complement. These findings are the first to demonstrate the participation of satDNA sequences in the karyotype diversification of wild diploid Arachis species.

Non-random chromosome arrangement in triploid endosperm nuclei

The endosperm is at the center of successful seed formation in flowering plants. Being itself a product of fertilization, it is devoted to nourish the developing embryo and typically possesses a triploid genome consisting of two maternal and one paternal genome complement. Interestingly, endosperm development is controlled by epigenetic mechanisms conferring parent-of-origin-dependent effects that influence seed development. In the model plant Arabidopsis thaliana, we have previously described an endosperm-specific heterochromatin fraction, which increases with higher maternal, but not paternal, genome dosage. Here, we report a detailed analysis of chromosomal arrangement and association frequency in endosperm nuclei. We found that centromeric FISH signals in isolated nuclei show a planar alignment that may results from a semi-rigid, connective structure between chromosomes. Importantly, we found frequent pairwise association of centromeres, chromosomal segments, and entire arms of chromosomes in 3C endosperm nuclei. These associations deviate from random expectations predicted by numerical simulations. Therefore, we suggest a non-random chromosomal organization in the triploid nuclei of Arabidopsis endosperm. This contrasts with the prevailing random arrangement of chromosome territories in somatic nuclei. Based on observations on a series of nuclei with varying parental genome ratios, we propose a model where chromosomes associate pairwise involving one maternal and one paternal complement. The functional implications of this predicted chromosomal arrangement are discussed.

An unusually high heterochromatin content and large genome size in the palm tree Trithrinax campestris (Arecaceae)

Trithrinax campestris (Burmeist.) Drude & Griseb., a neotropical member of tribe Cryosophileae, subfamily Coryphoideae (Arecaceae), forms small populations in the southern most extreme of the distribution for the tribe. To provide genetic information to assist its conservation, we performed detailed karyotype analysis in samples from Uruguay. The species displayed a karyotype with 2n = 36, a genome size (2C = 17.15 ± 0.07 pg) exceptionally high for a palm tree, and an elevated heterochromatin amount, represented by eight Chromomycin A3 (CMA) bands and numerous 4′,6-diamidino-2-phenylindole (DAPI) bands. 45S rDNA sites were found co-localising with CMA+ bands on Chromosome pairs 6, 9, 13 and 18, whereas a single pair of 5S rDNA sites was found on the proximal region of Chromosome pair 7. Its complex CMA and/or DAPI banding, together with the position of rDNA sites, allowed the identification of each chromosome pair and provided a useful tool to evaluate cytogenetic variations among populations and closely related species. Although this species maintains several karyotype similarities with its close relative T. brasiliensis, it can be promptly recognised by its DNA amount and DAPI bands. The large genome size in T. campestris is probably associated with its large blocks of heterochromatin, which represent 39% of its total chromosome length. Excluding the heterochromatin fraction, its chromosome size and 2C value become similar to those reported for most other diploid species in the family, suggesting that amplification of satellite DNA played an important role in its karyotype evolution.

Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics

To examine variation and taxonomic recognition of Pinus nigra (European black pine) at the intraspecific level, chromosomal distribution of 5S and 18S-5.8S-26S rDNA loci revealed by fluorescent in situ hybridisation (FISH) and fluorochrome banding with chromomycin A(3) and DAPI were analysed among allopatric populations belonging to different subspecies. Despite prevalent opinion on predominantly conserved and homogenous conifer karyotypes, several patterns were observed. Surprisingly, interstitial 18S rDNA loci and DAPI heterochromatin staining after FISH showed variations in distribution and localisation. Three subspecies shared a pattern with nine 18S rDNA loci (ssp. nigra, pallasiana and laricio) while ssp. dalmatica and salzmannii had eight rDNA loci. DAPI banding displayed two patterns, one with a high number of signals (ssp. nigra, pallasiana and dalmatica) and the other with a lower number of signals (ssp. salzmannii and laricio). We conclude that our results cannot provide proof for either classification scheme for the P. nigra complex, but rather demonstrate the variability of different heterochromatin fractions at the intraspecific level.

Morphometric and ultrastructural studies of the effect of infrared laser on rabbit temporo-mandibular joint fibroblasts

The infrared laser beams has been successfully used in joint lesions treatment due to its analgesic anti inflamatory and healing action, and on a cellular level, inducing an accentuated synthesis of ATP, activating cellular proliferation and collagen synthesis. Samples of normal-control and irradiated tissue from temporo-mandibular joint disc of rabbit, were obtained, with daily doses of 2 joules/cm²during 10 consecutive days. They were prepared for transmission electronic microscopy.Transmission electronic micrographs of normal and irradiated fibroblasts were obtained with final increase of 11.500 X for morphometric studies with the purpose of quantifying the volumetric fractions of the cellular components pertaining to the mentioned cellular types. Volumetric fractions of the quoted cell corresponding to nucleus, cytoplasm, rough endoplasmatic reticule, eu and heterochromatin were evaluated. In the same manner, the nucleus - cytoplasmatic relation and the area of each cellular type were quantified. The results derived from the morphometric comparative study between the normal and irradiated fibroblasts indicated that there were significant differences with respect to the volumetric fractions of euchromatin and heterochromatin and fundamentally in the cellular areas of both types. On the other hand, the rest of quantified parameters remained constant. It could be concluded that the cellular function reflected in collagen synthesis and secretion remained constant in both cellular types. However, by means of infrared laser stimulations, this fibroblast facilitated protein synthesis, due to high percentage of transcriptionally active euchromatine.

Cytogenetic mapping of common bean chromosomes reveals a less compartmentalized small-genome plant species

Cytogenetic maps of common bean chromosomes 3, 4 and 7 were constructed by fluorescence in-situ hybridization (FISH) of BAC and a few other genomic clones. Although all clones were selected with genetically mapped markers, mostly with single-copy RFLPs, a large subset of BACs, from 13 different genomic regions, contained repetitive sequences, as concluded from the regional distribution patterns of multiple FISH signals on chromosomes: pericentromeric, subtelomeric and dispersed. Pericentromeric repeats were present in all 11 chromosome pairs with different intensities, whereas subtelomeric repeats were present in several chromosome ends, but with different signal intensities depending on the BAC, suggesting that the terminal heterochromatin fraction of this species may be composed of different repeats. The correlation of genetic and physical distances along the three studied chromosomes was obtained for 23 clones. This correlation suggests suppression of recombination around extended pericentromeric regions in a similar way to that previously reported for plant species with larger genomes. These results indicate that a relatively small plant genome may also possess a large proportion of repeats interspersed with single-copy sequences in regions other than the pericentromeric heterochromatin and, nevertheless, exhibit lower recombination around the pericentromeric fraction of the genome.

Genome restructuring in mouse embryos during reprogramming and early development

Although a growing number of studies investigates functional genome organization in somatic cell nuclei, it is largely unknown how mammalian genome organization is established during embryogenesis. To address this question, we investigated chromocenter formation and the peculiar arrangements of chromosome domains in early mouse embryos. At the one-cell stage, we observed characteristic arrangements of chromosomes and chromocenter components. Subsequently, starting with the burst of zygotic genome transcription major rearrangements led to the establishment of somatic type chromocenters with a defined spatio-temporal organization. These processes appeared to be completed at the blastocyst stage with the onset of cell differentiation. During the same developmental period, a fraction of pericentric heterochromatin that was late replicating in the first cycle underwent switches in replication timing, spatial organization and epigenetic marks. Cloning experiments revealed that the genome organization typical for more advanced stages was quickly reverted into the one-cell stage-specific form after nuclear transfer, supporting the idea that reprogramming associated genome remodeling in normal and cloned embryos is determined by cytoplasmic factors. Together, the results suggest that distinct but characteristic forms of nuclear genome organization are required for genome reprogramming in early embryos and for proper regulation of differential gene expression patterns at later stages.

Dynamic Binding of the Human Telomeric Protein TRF1 to Intrachromosomal Blocks (TTAGGG)n in Live Chinese Hamster Cells Found to Depend on Transcription

Chromosomes of some vertebrates contain (TTAGGG)n tandem repeats not only in telomeres, but also in internal, often pericentric, regions. Such internal repeats, termed telomeric heterochromatin (THC), account for 5% of the genome in Chinese hamster. THC blocks dynamically bind the telomeric protein TRF1. The purpose of this study was to find out whether or not this interaction depends on transcription. Cells with normal transcription recovered approximately 85% of the initial fluorescence intensity (FI) of GFP-TRF1 60 s after photobleaching. The transcription inhibitor actinomycin D (ActD) quickly and almost completely suppressed GFP-TRF1 exchange (only 10% of the initial FI was recovered) when used at a concentration fully inhibiting DNA-dependent RNA polymerases I and II. The protein synthesis inhibitor cycloheximide had no such effect. The efficiency of FI recovery did not appreciably change when ActD was used at a low concentration to suppress only RNA polymerase I. Since some fractions of heterochromatin are actively transcribed in mammalian cells, it was assumed that GFP-TRF1 exchange is associated either with heterochromatin transcription, which is possibly necessary for siRNA synthesis and self-maintenance of heterochromatin, or with inhibited expression of other genes affecting the TRF1 stability.

Ribosomal DNA heterochromatin in plants

The aim of this review is to integrate earlier results and recent findings to present the current state-of-the-art vision concerning the dynamic behavior of the ribosomal DNA (rDNA) fraction in plants. The global organization and behavioral features of rDNA make it a most useful system to analyse the relationship between chromatin topology and gene expression patterns. Correlations between several heterochromatin fractions and rDNA arrays demonstrate the heterochromatic nature of the rDNA and reveal the importance of the genomic environment and of developmental controls in modulating its dynamics.

Characterization of restriction enzyme banding polymorphisms in human chromosomes.

C and Re-banding chromosome heteromorphisms were analysed in blood cultures from 43 normal individuals. Restriction enzymes used were AluI, DdeI, HaeIII, and MboI. Chromosome pairs exhibiting heteromorphisms were: 1, 3, 4, 6, 9, 10, 12-16, and 18-22. Each individual showed a specific combination of C- and Re-banding heteromorphisms not shared by any other individual in the series. Some polymorphisms could be detected by all the banding methods used. Others could be detected by some of the banding methods, and in some cases by only one of the banding methods used. The efficiency of each banding method to detect chromosomal polymorphisms depended on the type of polymorphism and the chromosomal pair analysed. Our results indicate that Re-banding polymorphisms occur due to changes in base composition of different fractions of heterochromatin or due to the presence or absence of different heterochromatic subsets. C- and Re-banding are complementary methods that expand the identification of chromosomal markers and which can be used to identify the parental origin of individual chromosomes.

Mitotic phosphorylation of histone H3 at threonine 3

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.

Phosphorylation of Histone H3 at Serine 10 Cannot Account Directly for the Detachment of Human Heterochromatin Protein 1γ from Mitotic Chromosomes in Plant Cells

Heterochromatin protein 1 (HP1) controls heterochromatin formation in animal cells, at least partly through interaction with lysine 9 (Lys-9)-methylated histone H3. We aimed to determine whether a structurally conserved human HP1 protein exhibits conserved heterochromatin localization in plant cells and studied its relation to modified histone H3. We generated transgenic tobacco plants and cycling cells expressing the human HP1gamma fused to green fluorescent protein (GFP) and followed its association with chromatin. Plants expressing GFP-HP1gamma showed no phenotypic perturbations. We found that GFP-HP1gamma is preferentially associated with the transcriptionally "inactive" heterochromatin fraction, a fraction enriched in Lys-9-methylated histone H3. During mitosis GFP-HP1gamma is detached from chromosomes concomitantly with phosphorylation of histone H3 at serine 10 and reassembles as cells exit mitosis. However, this phosphorylation cannot directly account for the dissociation of GFP-HP1gamma from mitotic chromosomes inasmuch as phosphorylation does not interfere with binding to HP1gamma. It is, therefore, possible that phosphorylation at serine 10 creates a "code" that is read by as yet an unknown factor(s), eventually leading to detachment of GFP-HP1gamma from mitotic chromosomes. Together, our results suggest that chromatin organization in plants and animals is conserved, being controlled at least partly by the association of HP1 proteins with methylated histone H3.

Histone H4 acetylation of euchromatin and heterochromatin is cell cycle dependent and correlated with replication rather than with transcription.

Reversible acetylation of nucleosomal histones H3 and H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains. Here, we report that the extent of H4 acetylation within euchromatin and heterochromatic domains is linked with DNA replication rather than with transcriptional activity, whereas H3 acetylation remains fairly constant throughout the cell cycle. Compared with euchromatin, plant nucleolus organizers were more strongly acetylated at H4 during mitosis but less acetylated during S phase, when the nucleolus appeared to be (at least transiently) devoid of nucleosomes. Deposition-related acetylation of lysines 5 and 12 of H4 seems to be conserved in animals and plants and extended to K16 in plants. A possibly species-specific above-average acetylation at lysines 9/18 and 14 of H3 appeared in 4',6-diamidino-2-phenylindole (DAPI)-stained heterochromatin fractions. These results were obtained by combining immunodetection of all acetylatable isoforms of H3 and H4 on mitotic chromosomes and nuclei in G1, early S, mid-S, late S, and G2 phases of the field bean with identification of specific chromatin domains by fluorescence in situ hybridization or DAPI staining. In addition, the histone acetylation patterns of distinct domains were compared with their replication and transcription patterns.

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.