Satellite DNA and heterochromatin variants: The case for unequal mitotic crossing over

Summary Variations of constitutive heterochromatin (heteromorphisms) appear to be a general feature of eucaryotes. A variety of molecular and cytogenetic evidence supports the hypothesis that heteromorphisms result from unequal double-strand exchanges during mitotic DNA replication. Constitutive heterochromatin consists of highly repeated DNA sequences that are not transcribed. Thus, heteromorphisms are tolerated without overt phenotypic effect. Several of the highly repeated DNAs that comprise constitutive heterochromatin have been shown to contain site-specific endonuclease recognition sequences interspersed at regular intervals dependent upon nucleosome structure. These interspersed short repeated sequences could mediate unequal crossovers, resulting in quantitative variability of constitutive heterochromatin and satellite DNA. De novo variations of constitutive heterochromatin may be useful as markers of exposure to mutagens and/or carcinogens.     

Translocation of unstable heterochromatin as the mechanism of sister chromatid exchange formation: a proposed hypothesis

The phenomenon of sister chromatid exchange has remained an enigma in that the actual mechanism for its formation has never been elucidated. It has long been suspected that the process involves some form of breakage and rejoining of DNA, but that hypothesis has never been proved. Recent work in this laboratory using cells from a premature aging disorder (Werner's syndrome) has promulgated the hypothesis that heterochromatin may not be an integral structure of chromosomes, but rather serves as a surface feature or covering. Furthermore, heterochromatin in Werner's syndrome chromosomes was found to be unstable and easily sloughed-off the chromosome surface. In this investigation evidence is presented which shows that incorporation of bromodeoxyuridine into DNA causes instability in the purported heterochromatin covering, resulting in translocation of segments of heterochromatin from the unifilarly-substituted chromatid to the bifilarly-substituted sister chromatid. Such translocation may represent the long-elusive mechanism of sister chromatid exchange formation.     

Cytological studies of heterochromatin function in the Drosophila melanogaster male: autosomal meiotic pairing

In Drosophila melanogaster it is now documented that the different satellite DNA sequences make up the majority of the centromeric heterochromatin of all chromosomes. The most popular hypothesis on this class of DNA is that satellite DNA itself is important to the pairing processes of chromosomes. Evidence in support of such a hypothesis is, however, circumstantial. This hypothesis has been evaluated by direct cytological examination of the meiotic behaviour of heterochromatically and/or euchromatically rearranged autosomes in the male. It was found that neither substantial deletions nor rearrangements of the autosomal heterochromatin cause any disruption of meiotic pairing. Autosomal pairing depends on homologs retaining sufficient euchromatic homology. This is the first clear demonstration that the highly repeated satellite DNA sequences in the heterochromatin of the second, third and fourth chromosomes are not important in meiotic pairing, but rather that some euchromatic homology in the autosomes is essential to ensure a regular meiotic process. These results on the autosomes, when taken in conjunction with our previous studies on sex chromosome pairing, clearly indicate that satellite DNA is not crucial for male meiotic chromosome pairing of any member of the D. melanogaster genome.     

Heterochromatin DNA replication and Rif1

Constitutive heterochromatin is essential for chromosome maintenance in all eukaryotes. However, the repetitive nature of the underlying DNA, the presence of very stable protein-DNA complexes and the highly compacted nature of this type of chromatin represent a challenge for the DNA replication machinery. Data collected from different model organisms suggest that at least some of the components of the DNA replication checkpoint could be essential for ensuring the completion of DNA replication in the context of heterochromatin. I review and discuss the literature that directly or indirectly contributes to the formulation of this hypothesis. In particular, I focus my attention on Rif1, a newly discovered member of the DNA replication checkpoint. Recent data generated in mammalian cells highlight the spatial and temporal relation between Rif1, pericentromeric heterochromatin and S-phase. I review these recent and the previous data coming from studies performed in yeast in order to highlight the possible evolutionary conserved links and propose a molecular model for Rif1 role in heterochromatin replication.     

In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.by D. Schweizer     

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.     

Hoechst 33258, distamycin A,and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.by E.R. Schmidt     

The origin of heterochromatin in eukaryotes

This study is an attempt to reconstruct the stages of the evolution of heterochromatin in eukaryotes. According to the hypothesis put forward in the work, the origin of satellite DNAs (stDNAs) was directly related to certain functional characteristics of DNA polymerases, and stDNAs themselves are products of accidental slippage at replication initiation sites. Even at the moment when the stDNAs precursors (protosatellites) appeared, they had properties of selfish DNA. Therefore, specific complex mechanisms of genetic control of their replication and recombination have developed in evolution to restrict the spread of these DNAs over the genome. The host control over protosatellites has led to the appearance of the main heterochromatic characteristics in them, such as late replication, decreased recombination, and denser chromatin packing compared to euchromatin. The next stage of heterochromatin evolution led to the union of protosatellite clusters and ordinary genes if late replication was necessary for these genes or if gene complexes already formed required protection from the destructure effect of crossing over. The known cases of location of certain genes in heterochromatic blocks in Drosophila melanogaster, the eukaryote that has been best studied genetically, confirm this hypothesis.     

Interchromosomal Effects of Heterochromatic Deletions on Recombination in DROSOPHILA MELANOGASTER

It is now known that partial deletions of the satellite sequences in X-chromosome heterochromatin result in a significant decrease in intrachromosomal recombination in the proximal region of the X chromosome of D. melanogaster (YAMAMOTO and MIKLOS 1978). It is important to ask then if the loss or gain of heterochromatin on the X also alters recombination in other chromosomes of the genome (interchromosomal effects). I have looked for such alterations by measuring recombination in chromosome 3. The results clearly indicate that the partial loss of X-chromosome heterochromatin not only decreases crossing over in the proximal region of the X chromosome itself, but also increases the frequency in chromosome 3, especially in the euchromatic regions around the centromere. Furthermore, the greater the deficiency of X heterochromatin, the higher is recombination in chromosome 3. This finding not only provides further evidence in support of the hypothesis that heterochromatin, in this case mainly composed of satellite DNA, regulates the recombination system, but it demonstrates that when the satellite content of one chromosome of the D. melanogaster genome is altered, there is an alteration in the crossover characteristics of other chromosomes in the same complement. If the amount of satellite DNA in a genome is being continuously altered, then one can predict that the recombination system is also being continually perturbed. Thus, the changing gene combinations produced indirectly by increases or decreases of heterochromatin are among the components available to organisms to break up or form new gene combinations upon which selection can act.     

Islands of Complex DNA Are Widespread in Drosophila Centric Heterochromatin

Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three ``islands' of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.     

The Origin of Heterochromatin in Eukaryotes

This study is an attempt to reconstruct the stages of the evolution of heterochromatin in eukaryotes. According to the hypothesis put forward in the work, the origin of satellite DNAs (stDNAs) was directly related to certain functional characteristics of DNA polymerases, and stDNAs themselves are products of accidental slippage at replication initiation sites. Even at the moment when the stDNAs precursors (protosatellites) appeared, they had properties of selfish DNA. Therefore, specific complex mechanisms of genetic control of their replication and recombination have developed in evolution to restrict the spread of these DNAs over the genome. The host control over protosatellites has led to the appearance of the main heterochromatic characteristics in them, such as late replication, decreased recombination, and denser chromatin packing compared to euchromatin. The next stage of heterochromatin evolution led to the union of protosatellite clusters and ordinary genes if late replication was necessary for these genes or if gene complexes already formed required protection from the destructure effect of crossing over. The known cases of location of certain genes in heterochromatic blocks in Drosophila melanogaster,the eukaryote that has been best studied genetically, confirm this hypothesis.     

The distribution of repetitive DNA in the chromosomes of cultured cells of Drosophila melanogaster

The distribution patterns of different stains (orcein, quinacrine and Giemsa) in an established cell line of Drosophila melanogaster (GM₃ WS) were compared. Each chromosome stained both with quinacrine and with Giemsa shows up a specific banding pattern for heterochromatin. The comparison between the two patterns suggests a hypothesis concerning the significance of the fluorescence; moreover it permits the conclusion that heterochromatin in D. melanogaster mitotic chromosomes is all constitutive and that there is a correspondence between repetitive DNA and sections poor in mappable genes.This work was supported by a grant of the Consiglio Nazionale delle Ricerche Roma.     

Replication of heterochromatin: insights into mechanisms of epigenetic inheritance

Heterochromatin is composed of tightly condensed chromatin in which the histones are deacetylated and methylated, and specific nonhistone proteins are bound. Additionally, in vertebrates and plants, the DNA within heterochromatin is methylated. As the heterochromatic state is stably inherited, replication of heterochromatin requires not only duplication of the DNA but also a reinstallment of the appropriate protein and DNA modifications. Thus replication of heterochromatin provides a framework for understanding mechanisms of epigenetic inheritance. In recent studies, roles have been identified for replication factors in reinstating heterochromatin, particularly functions for origin recognition complex, proliferating cell nuclear antigen, and chromatin-assembly factor 1 in recruiting the heterochromatin binding protein HP1, a histone methyltransferase, a DNA methyltransferase, and a chromatin remodeling complex. Potential mechanistic links between these factors are discussed. In some cells, replication of the heterochromatin is blocked, and in Drosophila this inhibition is mediated by a chromatin binding protein SuUR.     

Clonal variants of constitutive heterochromatin of human fibroblasts after recovery from mitomycin treatment

Presumptive clones of human skin fibroblast-like cells surviving mitomycin C treatments show a variety of intra- and interchromosomal rearrangements limited to the constitutive heterochromatic regions. Starting with a wild-type line polymorphic for chromosome no. 1 heterochromatin, we have observed clones with complete symmetry and varying degrees of asymmetry of the no. 1 heterochromatin, translocations of excess chromosome 1 heterochromatin to one no. 9 member, interstitial translocations to sites normally devoid of heterochromatin, and duplication of the Y chromosome long arm heterochromatin. In the case of the chromosome no. 1 pair, the extent of heterochromatin variation was quantitated to test the hypothesis that discrete classes of variants occur. There appear to be two types of variants: Those showing reciprocal changes between homologues (2 examples) and those showing a change in the amount of heterochromatin of a single homologue (5 examples). The latter group showed an approximately linear series of variants. Unequal cross-over following repair after damage from the alkylating agent is considered the most likely explanation for the observed changes, given the repetitive nature of DNA at these heterochromatic sites.     

Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential

Rearrangements in heterochromatin in the vicinity of the centromeres of chromosomes 1 and 16 are frequent in many types of cancer, including ovarian epithelial carcinomas. Satellite 2 DNA is the main sequence in the unusually long heterochromatin region adjacent to the centromere of each of these chromosomes. Rearrangements in these regions and hypomethylation of satellite 2 DNA are a characteristic feature of patients with a rare recessive genetic disease, ICF (immunodeficiency, centromeric region instability, and facial anomalies). In all normal tissues of postnatal somatic origin, satellite 2 DNA is highly methylated. We examined satellite 2 DNA methylation in ovarian tumors of different malignant potential, namely, ovarian cystadenomas, low malignant potential (LMP) tumors, and epithelial carcinomas. Most of the carcinomas and LMP tumors exhibited hypomethylation in satellite 2 DNA of both chromosomes 1 and 16. A comparison of methylation of these sequences in the three types of ovarian neoplasms demonstrated that there was a statistically significant correlation between the extent of this satellite DNA hypomethylation and the degree of malignancy (P<0.01). Also, there was a statistically significant association (P<0.005) between genome-wide hypomethylation and undermethylation of satellite 2 DNA among these 17 tumors. In addition, we found abnormal hypomethylation of satellite alpha DNA in the centromere of chromosome 1 in many of these tumors. Our findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypomethylation which might predispose cells to structural and numerical chromosomal aberrations. Several of the proteins that bind to pericentromeric heterochromatin are known to be sensitive to the methylation status of their target sequences and so could be among the sensors for detecting abnormal demethylation and mediating effects on chromosome structure and stability.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Perinuclear DNA and heterochromatin

We have analyzed how and to what measure DNA which replicates specifically at the nuclear membrane at the end of S phase (perinuclear DNA) correlates with heterochromatin. We found that, although perinuclear DNA can be distinguished in autoradiographs in two different patterns of labelling, these appear to constitute the same heterochromatic sites. Furthermore, since perinuclear DNA is only replicated in 7% of the whole S phase, while DNA in heterochromatin amounts to about 25–30% of all chromatin, we concluded that perinuclear DNA only occurs in a small fraction of all heterochromatin.     

Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.     

The response to DNA damage in heterochromatin domains

Eukaryotic genomes are organized into chromatin, divided into structurally and functionally distinct euchromatin and heterochromatin compartments. The high level of compaction and the abundance of repeated sequences in heterochromatin pose multiple challenges for the maintenance of genome stability. Cells have evolved sophisticated and highly controlled mechanisms to overcome these constraints. Here, we summarize recent findings on how the heterochromatic state influences DNA damage formation, signaling, and repair. By focusing on distinct heterochromatin domains in different eukaryotic species, we highlight the heterochromatin contribution to the compartmentalization of DNA damage repair in the cell nucleus and to the repair pathway choice. We also describe the diverse chromatin alterations associated with the DNA damage response in heterochromatin domains and present our current understanding of their regulatory mechanisms. Finally, we discuss the biological significance and the evolutionary conservation of these processes.