[Polycomb and trithorax control genome expression by determining the alternative epigenetic states of chromatin for key developmental regulators].

The Polycomb (PcG) and Trithorax (TrxG) group proteins are essential for development in all multicellular organisms. Mutations of the PcG and TrxG genes act as early embryonic lethals, while their overexpression correlates with malignancies. Comparative genome analysis showed that PcG and TrxG form a binary regulatory system that functions as an epigenetic rheostat to determine the threshold levels of extracellular signals affecting the expression levels of key developmental genes.

Polycomb silencing mechanisms and genomic programming.

Polycomb complexes, best known for their role in the epigenetic silencing of homeotic genes, are now known to regulate a large number of functions in organisms from flies to man. They control transcription activators, pattern-forming genes, maintenance of stem cells and are implicated in cell proliferation and oncogenesis. Our understanding of Polycomb mechanisms derives principally from the study of homeotic genes in Drosophila, where they act in an all-or-none fashion to silence expression in inappropriate parts of the organism. This review summarizes what has been learned from homeotic genes and examines the possible extensions of Polycomb mechanisms to allow for dynamic regulatory behavior and the reprogramming of silenced chromatin states.

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication.

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR- phenotype and induces specific swellings in heterochromatin.

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.

The Drosophila suppressor of underreplication protein binds to late-replicating regions of polytene chromosomes.

In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin.

Establishment of Polycomb silencing requires a transient interaction between PC and ESC.

Two distinct types of Polycomb complexes have been identified in flies and in vertebrates, one containing ESC and one containing PC. Using LexA fusions, we show that PC and ESC can establish silencing of a reporter gene but that each requires the presence of the other. In early embryonic extracts, we find PC transiently associated with ESC in a complex that includes EZ, PHO, PH, GAGA, and RPD3 but not PSC. In older embryos, PC is found in a complex including PH, PSC, GAGA, and RPD3, whereas ESC is in a separate complex including EZ, PHO, and RPD3.

Painting of fourth, a chromosome-specific protein in Drosophila.

Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila melanogaster, a complex including the five Male-Specific Lethal (MSL) proteins, "paints" the male X chromosome, mediating its hypertranscription. Here, with the molecular cloning of Painting of fourth (Pof), we describe a previously uncharacterized gene encoding a chromosome-specific protein in Drosophila. Unlike the MSL proteins, POF paints an autosome, the fourth chromosome of Drosophila melanogaster. Chromosome translocation analysis shows that the binding depends on an initiation site in the proximal region of chromosome 4 and spreads in cis to involve the entire chromosome. The spreading depends on sequences or structures specific to chromosome 4 and cannot extend to parts of other chromosomes translocated to the fourth. Spreading can also occur in trans to a paired homologue that lacks the initiation region. In the related species Drosophila busckii, POF paints the entire X chromosome exclusively in males, suggesting relationships between the fourth chromosome and the X and between POF complexes and dosage-compensation complexes.

Loss of insulator activity by paired Su(Hw) chromatin insulators.

Chromatin insulators are regulatory elements that block the action of transcriptional enhancers when interposed between enhancer and promoter. The Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insulator and prevents enhancer-promoter interaction by a mechanism that is not understood. We show that when two copies of the Su(Hw) insulator element, instead of a single one, are inserted between enhancer and promoter, insulator activity is neutralized and the enhancer-promoter interaction may instead be facilitated. This paradoxical phenomenon could be explained by interactions between protein complexes bound at the insulators.

Recruitment of components of Polycomb Group chromatin complexes in Drosophila.

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, we have targeted various Polycomb Group (PcG) proteins by fusing them to the LexA DNA binding domain. When LexA-PC, -PSC, -PH or -SU(Z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-PHO is neither able to silence nor to interact with PC-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose PHO-binding sites are mutated suggests that, while PHO is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing.

[Effect of four doses of the Su(UR)ES gene on intercalary heterochromatin in Drosophila melanogaster]. / Effekt chetyrekh doz gena Su(UR)ES na interkaliarnyi geterokhromatin u Drosophila melanogaster.

Polytene chromosomes of salivary glands of various Drosophila melanogaster strains containing two doses of the normal Su(UR)ES allele have a constant set of intercalary heterochromatin (IHC) sites. Their DNA is underreplicated, which leads to breaks and ectopic contacts emerging at a certain rate. Almost no underreplication, breaks, or ectopic conjugation are present in mutants lacking the normal Su(UR)ES gene product. It could be expected that an increase in the number of the Su(UR)ES+ gene doses would, in turn, drastically increase ectopic conjugation and breakage. To test this hypothesis, a strain of D. melanogaster was obtained with two additional doses of Su(UR)ES+ introduced into its genome. The flies with four gene doses exhibited a considerable increase in ectopic conjugation: both the proportion of regions participating in conjugation and the number of chromosomes with numerous contact nodes were increased. As a result, chromosomes that were straight and well-stretched in homozygotes for the mutation in Su(UR)ES became twisted and wound and contained many loops or nodes. Many chromosomes were wound too tightly for cytological analysis. Four doses of Su(UR)ES+ considerably increased the number of weak "points." For example, the 2R chromosome has only 3 weak points in strains with two doses of Su(UR)ES+ and as many as 22 weak points in the strain with four doses. In the transgenic strain, the frequency of breaks in previously known weak points increased, and new breaks appeared in 19 additional sites. All new break points appeared in the regions that were earlier described as regions of late replication in the S phase.

Structure of a polycomb response element and in vitro binding of polycomb group complexes containing GAGA factor.

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.

Detection of neuroblastoma in the bone marrow: biopsy versus aspiration.

PURPOSE: Multiple studies have emphasized the higher yield of detection of metastatic neuroblastoma (MNb) by bone marrow biopsy (BMB) than by bone marrow aspiration (BMA). Because the need for BMA has been questioned, the yield of both procedures was investigated at diagnosis and during the course of disease. METHODS: For morphologic and immunohistochemical detection of MNb, 289 specimens obtained by BMA and BMB from 57 children with neuroblastoma were reviewed. RESULTS: In 34% of cases, MNb was present in both the aspirate and biopsy specimen. MNb was present in only the biopsy specimen in 8% and in only the aspirate in 6%. In 52%, neither BMA nor BMB detected MNb. In 15 of 18 cases in which MNb was present in the aspirate only, protein gene product 9.5 (PGP) stain was performed on the biopsy specimen. In one case, this helped to identify MNb that was not evident by routine hematoxylin and eosin stain. Of the 24 cases in which only the BMB was positive, 3 were identified only by means of PGP stain. CONCLUSIONS: Even with the additional use of immunohistochemistry, both BMA and BMB should be performed to have the highest yield of detection of MNb in bone marrow.

Chromatin insulator elements block the silencing of a target gene by the Drosophila polycomb response element (PRE) but allow trans interactions between PREs on different chromosomes.

Polycomb response elements (PREs) can establish a silenced state that affects the expression of genes over considerable distances. We have tested the ability of insulator or boundary elements to block the repression of the miniwhite gene by the Ubx PRE. The gypsy element and the scs element interposed between PRE and miniwhite gene protect it against silencing but the scs' is only weakly effective. When the PRE-miniwhite gene construct is insulated from flanking chromosomal sequences by gypsy elements at both ends, it can still establish efficient silencing in some lines but not others. We show that this can be caused by interactions in trans with PREs at other sites. PRE-containing transposons inserted at different sites or even on different chromosomes can interact, resulting in enhanced silencing. These trans interactions are not blocked by the gypsy insulator and reveal the importance of nonhomologous associations between different regions of the genome for both silencing and activation of genes. The similarity between the behavior of PREs and enhancers suggests a model for their long-distance action.

Chromatin-silencing mechanisms in Drosophila maintain patterns of gene expression.

The Polycomb-Group proteins form chromatin complexes that can silence gene expression over large distances. The formation of these complexes at homeotic genes depends on early developmental events but the repressed state is then maintained through many cell divisions. In vivo, complexes formed at one genomic site can interact with those at other sites, suggesting that they, like heterochromatin complexes, affect the folding of chromatin and the organization of chromosomes in the nucleus.

PcG complexes and chromatin silencing.

Silencing complexes in yeast and in the fly have many similarities. This repressive complex is assembled by a chain of recruitment; its extent and stability depend on the concentration of components and affect an extended chromatin region, probably through interactions with nucleosomes. Recent results show that assembly of the complex is antagonized by transcriptional activity in the region but is favored by interactions with other complexes nearby or in other regions that associate in the same nuclear environment. How such a complex interferes with transcriptional activity is not entirely clear but current evidence suggests that they compete with the chromatin structure required for the binding of activators.