[Position Effect Variegation: Role of the Local Chromatin Context in Gene Expression Regulation].

Position effect variegation (PEV) is a phenomenon wherein the expression level of a gene strongly depends on its genomic position. PEV can be observed when a gene is moved via a chromosome rearrangement or identical genetic constructs are inserted into different regions of the genome. The eukaryotic genome has a domain organization, and gene activity within a domain depends not only on the nucleotide sequence of a gene, but also on the state of surrounding chromatin, thus being regulated epigenetically. Chromatin is a complex of DNA, RNA, and associated structural and regulatory proteins. The epigenetic status of chromatin depends on the replication time of a given genomic region, particular regulatory DNA motifs, and contacts with the inner nuclear envelope (lamina) and other chromosome regions (topologically associated domains). PEV results from the changes in the epigenetic state of a gene and provides a unique tool to study the molecular and biochemical processes that underlie the establishment and switching of epigenetic states. Understanding the molecular mechanisms of PEV in human is of clinical importance, in particular, for the detection and treatment of retroviral infections because the local chromatin state may determine the latent/active state transition of an infection, such as HIV. In addition, a large number of human neurodegenerative diseases are caused by epigenetic gene inactivation due to expansion of short repeats. Finally, to apply gene therapy methods, it is important to develop approaches that ensure a necessary level of transgene expression with sufficient accuracy.

PATHWAYS OF SPINDLE FORMATION IN DROSOPHILA MITOTIC AND MEIOTIC CELLS.

Spindle assembly relies on three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs nucleated near the chromosomes/kinetochores and MTs nucleated from preexisting MTs through the augmin-based pathway. Here, we review the roles of these microtubule generation pathways in Drosophila spindle assembly. The extant results indicate that female meiotic cells, male meiotic cells, larval brain cells and S2 tissue culture cells exploit specific pathway combinations for generating the MTs necessary for spindle formation. Thus, different Drosophila cell types have specific modes of spindle assembly, which might be related to specific functional and developmental requirements.

Local DNA underreplication correlates with accumulation of phosphorylated H2Av in the Drosophila melanogaster polytene chromosomes.

DNA in Drosophila melanogaster polytene chromosomes is known to be locally underreplicated in both pericentric and intercalary heterochromatin. When the SuUR gene is mutant, complete and partial suppression of underreplication are observed in intercalary and pericentric heterochromatin, respectively; in contrast, overexpression of SuUR results in stronger underreplication. Using antibodies against phosphorylated histone H2Av and flies with different levels of SuUR expression, we demonstrated a clear correlation between the extent of underreplication in specific chromosome regions and the accumulation of H2Av phosphorylated at S137 (gamma-H2AX) at the same sites. Phosphorylated H2Av is a well-established marker of DNA double-stranded breaks (DSB). Our data thus argue that DNA underreplication leads to DSBs and that DSBs accumulate as salivary gland cells progress throughout repeated endocycles. We speculate that ligation of free double-stranded DNA termini causes the formation of ectopic contacts between the underreplicated regions in heterochromatin.

Intercalary heterochromatin in polytene chromosomes of Drosophila melanogaster.

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.

Polytene chromosomes: 70 years of genetic research.

Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and position effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model.

Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes.

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.