Repeated, protein-encoding heterochromatic genes cause inactivation of a juxtaposed euchromatic gene

Euchromatic genes are often silenced by rearrangements that place them within or near heterochromatin, a phenomenon known as position effect variegation (PEV). However, little is known about molecular structure of cis-acting heterochromatic fragments responsible for PEV. Here we report that heterochromatic cluster containing Stellate repeats, that encode putative regulatory subunit of protein kinase CK2 cause PEV of a reporter white 'mini-gene'. It is the first example of an euchromatic gene being silenced because of the proximity to the natural, well-defined heterochromatic repeat cluster.     

Treatment of temporomandibular disorders: arthroscopy, orthodontics, kinesitherapy]

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A-2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes.     

Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.     

Genetic modification of heterochromatic association and nuclear organization in Drosophila

Heterochromatin is the highly compact, usually pericentromeric, region of eukaryotic chromosomes. Unlike the more gene-rich euchromatin, heterochromatin remains condensed during interphase, when it is sequestered to the periphery of the nucleus. Here we show, by using fluorescent in situ hybridization to interphase diploid nuclei of Drosophila, that the insertion of heterochromatin into a euchromatic gene, which results in position-effect variegation (PEV), also causes the aberrant association of the gene and its homologous copy with heterochromatin. In correlation with the gene's mutant variegating phenotype, the cytological association of the heterochromatic region is affected by chromosomal distance from heterochromatin and by genic modifiers of PEV. Proteins that are thought to be involved in the formation of heterochromatin can therefore influence the interphase nuclear position of a chromosomal region. This suggests that heterochromatin and proteins involved in its formation provide a structural framework for the interphase nucleus.     

In vivo analysis of scaffold-associated regions in Drosophila: a synthetic high-affinity SAR binding protein suppresses position effect variegation

Scaffold-associated regions (SARs) were studied in Drosophila melanogaster by expressing a synthetic, high-affinity SAR-binding protein called MATH (multi-AT-hook), which consists of reiterated AT-hook peptide motifs; each motif is known to recognize a wide variety of short AT-rich sequences. MATH proteins were expressed specifically in the larval eye imaginal discs by means of the tetracycline-regulated transactivation system and tested for their effect on position effect variegation (PEV). MATH20, a highly potent SAR ligand consisting of 20 AT-hooks, was found to suppress whitemottled 4 variegation. This suppression required MATH20 expression at an early larval developmental stage. Our data suggest an involvement of the high AT-rich SARs in higher order chromatin structure and gene expression.     

The effect of nitrate and nitrite on the respiration and cytochrome system of Rhizobium lupini]

Chromosome replication has been analysed in four species of Chilocorus. In C. ORBUS Csy., C. tricyclus Smith, and C. hexacyclus Smith, centric regions of all chromosomes are last to replicate, preceded in order by heterochromatic arms and euchromatic arms. In C. stigma Say, very late replication of centric regions can be detected only in otherwise wholly euchromatic chromosomes (= monophasics); in chromosomes with one arm heterochromatic ( = disphasics), these arms are last to replicate. Based on pachytene bivalent morphology and chromosome banding patterns, and supported by autoradiographic data, models are presented for the general organisation of Chilocorus chromosomes. All chromosomes in the first three species are subdivided into euchromatic arm, centric heterochromatin, and either a second euchromatic are (monophasics) or a heterochromatic arm (diphasics). Chilocorus stigma diphasics apparently lack distinct centric organisation, and are therefore divided into euchromatic and heterochromatic arms only.     

A position effect variegation model of chromatin compaction

A position effect variegation (PEV) model of chromatin compaction is proposed, based on the concept of a statistical distribution of compaction protein (CP) molecules around compaction initiation centers (CICs). The principles of the model are as follows: (1) CICs are present in both hetero- and euchromatin, and (2) different CP molecules interact not only with DNA but also with one another, forming a multimeric complex. When a certain level of DNA-protein binding is exceeded, heterochromatic domains are formed. The model suggests that continuous and discontinuous chromatin compaction resulting from PEV is due to an irregular CIC distribution along the chromosome.     

The product of proliferation disrupter is concentrated at centromeres and required for mitotic chromosome condensation and cell proliferation in Drosophila

Homozygosity for a null mutation in the proliferation disrupter (prod) gene of Drosophila causes decreased mitotic index, defects of anaphase chromatid separation, and imperfect chromosome condensation in larval neuroblasts and other proliferating cell populations. The defective condensation is especially obvious near the centromeres. Mutant larvae show slow growth and massive cell death in proliferating cell populations, followed by late larval lethality. Loss of prod function in mitotic clones leads to the arrest of oogenesis in the ovary and defective cuticle formation in imaginal disc derivatives. The prod gene encodes a novel 301-amino-acid protein that is ubiquitously expressed and highly concentrated at the centric heterochromatin of the second and third mitotic chromosomes, as well as at > 400 euchromatic loci on polytene chromosomes. We propose that Prod is a nonhistone protein essential for chromosome condensation and that the chromosomal and developmental defects are caused by incomplete centromere condensation in prod mutants.     

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression

The position effect of the cubitus interruptus (ci) gene occurs when this gene, which is normally located in the vicinity of the pericentric heterochromatin of chromosome 4, is transferred by chromosome rearrangements to euchromatin regions. Cytological aspects of this phenomenon were investigated. For six reciprocal translocations causing the position effect (Dubinin effect) of ci, the frequencies of the ectopic contacts of the translocated chromosome 4 homologue with pericentric heterochromatin were compared to the conjugation frequencies of this chromosome's homologues. The frequencies were significantly higher when the gene was transferred to proximal chromosome regions. This suggested that the suppression of the Dubinin effect in the case of translocations with euchromatin breaks in proximal chromosome regions is caused by the higher conjugation frequency of translocated and normal chromosome 4 homologues in proximal than in distal regions. The effect of genes modulo and Su(var)2-05, which are known as modifiers of the position effect variegation, on the conjugation frequency of chromosome 4 homologues was studied for three translocations. It was shown that modulo did not affect this frequency, whereas Su(var)205 significantly decreased it. Cytogenetic data confirmed the association of the ci position effect with damage in the somatic pairing of chromosome 4 homologues. These data indicate that pericentric heterochromatin participates in determination of the localization of chromosome regions in the interphase nucleus.     

Dynamic organization of the beta-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25 degrees C and beta-heterochromatic in X0 males at 14 degrees C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(ILR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

The relationship of a specific chromosomal region to the development of the acrosome

In early spermatids of Urodeles the chromosome segments bearing constitutive heterochromatin are localized in one half of the round nucleus; this region becomes the basal part of the long nucleus of the spermatozoon. The euchromatic chromosome segments extend toward the anterior nuclear pole in a bouquet configuration (Macgregor and Walker, 1973). In the course of spermiohistogenesis, one of the heterochromatic regions (the acrosomal chromocenter) migrates from the basal part to the anterior half of the spermatid nucleus. This heterochromatic block is identical with a species-specific, definite C-band in the karyotype. This relationship between the acrosomal chromocenter and a specific chromosomal C-band was established in Triturus cristatus, T. marmoratus, T. alpestric and Cynops pyrrhogaster. In closely related species this particular C-band lies on similar chromosomes. - While the spermatid nucleus still retains its round shape the acrosomal chromocenter despiralizes into a long heterochromatic thread (acrosomal thread). Precisely at the position of this thread the nucleus evaginates and acquires a pear-like shape. During the elongation of the nuclear protrusion the acrosomal thread remains associated with the anterior end. At termination of spermiogenesis it lies closely below the acrosome in the tip of the spermatozoon. Spontaneous aberrations which affect the acrosomal chromocenter or the thread lead to the development of spermatozoa with defective tips. - Several euchromatic segments, interspersed between the heterchromatic segments, can be recognized in the completely despiralized acrosomal thread. Genes responsible for the morphogenetic activities of both, the acrosomal chromocenter and the acrosomal thread, in the development of the spermtip, might be localized in these interspersed euchromatic segments. The existence in higher vertebrates of an acrosomal chromocenter or an equivalent chromosomal region is discussed.     

Hypomethylation of human sperm pronuclear chromosomes

Using the methylase SssI enzyme, we have analyzed the degree of in situ methylation of human sperm pronuclear chromosomes obtained by fertilizing hamster oocytes with human sperm. Untreated (control) sperm chromosome complements showed a higher degree of in situ methylation, compared to sperm complements previously treated with 5-azadeoxycytidine or lymphocyte chromosomes. This indicates that human sperm pronuclear chromosomes have a lower degree of genomic methylation compared to that of other somatic cells. The similarity in the degree of in situ methylation of the euchromatic and heterochromatic regions of chromosomes 1, 9, 15, and 16 and the Y chromosome in human sperm does not support the existence of a possible correlation between hypomethylation and heterochromatin decondensation.     

The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals

The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism. The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group. Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B. However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes. E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine. E(Pc) is expressed ubiquitously in Drosophila embryogenesis. E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites. Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter. This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function. Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans. Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C. elegans and humans. The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.     

Progression of atypical ductal hyperplasia/carcinoma in situ of the pancreas to invasive adenocarcinoma

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation.     

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.