Drosophila melanogaster as a model for studying protein-encoding genes that are resident in constitutive heterochromatin

The organization of chromosomes into euchromatin and heterochromatin is one of the most enigmatic aspects of genome evolution. For a long time, heterochromatin was considered to be a genomic wasteland, incompatible with gene expression. However, recent studies--primarily conducted in Drosophila melanogaster--have shown that this peculiar genomic component performs important cellular functions and carries essential genes. New research on the molecular organization, function and evolution of heterochromatin has been facilitated by the sequencing and annotation of heterochromatic DNA. About 450 predicted genes have been identified in the heterochromatin of D. melanogaster, indicating that the number of active genes is higher than had been suggested by genetic analysis. Most of the essential genes are still unknown at the molecular level, and a detailed functional analysis of the predicted genes is difficult owing to the lack of mutant alleles. Far from being a peculiarity of Drosophila, heterochromatic genes have also been found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Oryza sativa and Arabidopsis thaliana, as well as in humans. The presence of expressed genes in heterochromatin seems paradoxical because they appear to function in an environment that has been considered incompatible with gene expression. In the future, genetic, functional genomic and proteomic analyses will offer powerful approaches with which to explore the functions of heterochromatic genes and to elucidate the mechanisms driving their expression.     

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.     

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.     

Intercalary heterochromatin in polytene chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

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.     

Heterochromatin structure and function

Although heterochromatin has long been used as a model for studying chromatin condensation and heritable gene silencing, it is only relatively recently that detailed information has become available on the mechanisms that underlie its structure. Current evidence suggests that these operate on at least three different levels. A regular nucleosome array may facilitate packaging of the chromatin into a highly condensed configuration. Methylation of histone H3 lysine 9 and lysine 27 generates heterochromatin marks that are recognised through binding of heterochromatin proteins such as HP1. Finally, very recent studies using genetic and biochemical approaches have indicated that the RNAi machinery plays an important role in the formation of heterochromatin.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Genome‐wide distribution and functions of the AAE complex in epigenetic regulation in Arabidopsis

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1‐AIPP1‐EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin‐containing genes. However, the genome‐wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome‐wide level and preferentially interacts with genetic heterochromatin. Polyadenylation (poly(A) sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin‐containing genes, including not only intronic heterochromatin‐containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin‐overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin‐containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation.     

Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction

Genomic sequences provide powerful new tools in genetic analysis, making it possible to combine classical genetics with genomics to characterize the genes in a particular chromosome region. These approaches have been applied successfully to the euchromatin, but analysis of the heterochromatin has lagged somewhat behind. We describe a combined genetic and bioinformatics approach to the base of the right arm of the Drosophila melanogaster second chromosome, at the boundary between pericentric heterochromatin and euchromatin. We used resources provided by the genome project to derive a physical map of the region, examine gene density, and estimate the number of potential genes. We also carried out a large-scale genetic screen for lethal mutations in the region. We identified new alleles of the known essential genes and also identified mutations in 21 novel loci. Fourteen complementation groups map proximal to the assembled sequence. We used PCR to map the endpoints of several deficiencies and used the same set of deficiencies to order the essential genes, correlating the genetic and physical map. This allowed us to assign two of the complementation groups to particular "computed/curated genes" (CGs), one of which is Nipped-A, which our evidence suggests encodes Drosophila Tra1/TRRAP.     

Cytogenetic analysis of the third chromosome heterochromatin of Drosophila melanogaster

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49-h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47-h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.     

The effect of X chromosome heterozygosity on the structure of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA isolated from detergent-pronase lysates of adult flies has been used to look for ribosomal genes not integrated into the DNA of the chromosome in genotypes containing various combinations of inversions having breakpoints in the proximal heterochromatin of the X chromosome. Unintegrated genes are found in females heterozygous for inversions which have one breakpoint between the nucleolus organizer and the centromere. Homozygotes and males do not have unintegrated genes. The results suggest that unintegrated ribosomal genes result from an interaction between homologues having different arrangements of the proximal heterochromatin. In addition, data from a series of stocks carrying duplications of the X heterochromatin provide independent evidence for the size of the DNA on our gradients.     

Oxymoron no more: the expanding world of heterochromatic genes

Heterochromatin has been oversimplified and even misunderstood. In particular, the existence of heterochromatic genes is often overlooked. Diverse types of genes reside within regions classified as constitutive heterochromatin and activating influences of heterochromatin on gene expression in Drosophila are well documented. These properties are usually considered paradoxical because heterochromatin is commonly portrayed as "silent chromatin". In the past, studies of heterochromatic genes were limited to a few Drosophila genes. However, the recent discovery of several hundred heterochromatic genes in Drosophila, plants and mammals through sequencing projects offers new opportunities to examine the variety of ways in which heterochromatin influences gene expression. Comparative genomics is revealing diverse origins of heterochromatic genes and remarkable evolutionary fluidity between heterochromatic and euchromatic domains. These features justify a broader view of heterochromatin, one that accommodates repressive, permissive and activating effects on gene expression, and recognizes chromosomal and evolutionary transitional states between heterochromatin and euchromatin.     

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.     

Essential Genes in Autosomal Heterochromatin of Drosophila Melanogaster

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophilaheterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such ‘resident’ genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

FISH analysis of<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> heterochromatin using BACs and<Emphasis Type="Italic"> P</Emphasis> elements

The heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster contains about 30 essential genes defined by genetic analysis. In the last decade only a few of these genes have been molecularly characterized and found to correspond to protein-coding genes involved in important cellular functions. Moreover, several predicted genes have been identified by annotation of genomic sequence that are associated with polytene chromosome divisions 40, 41 and 80 but their locations on the cytogenetic map of the heterochromatin are still uncertain. To expand our current knowledge of the genetic functions located in heterochromatin, we have performed fluorescence in situ hybridization (FISH) mapping to mitotic chromosomes of nine bacterial artificial chromosomes (BACs) carrying several predicted genes and of 13 P element insertions assigned to the proximal regions of 2R and 3L. We found that 22 predicted genes map to the h46 region of 2R and eight map to the h47 regions of 3L. This amounts to at least 30 predicted genes located in these heterochromatic regions, whereas previous studies detected only seven vital genes. Finally, another 58 genes localize either in the euchromatin-heterochromatin transition regions or in the proximal euchromatin of 2R and 3L. Edited by: B. McKeeN. Corradini and F. Rossi contributed equally to this work     

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.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.     

A structural basis for variegating position effects

Variegating position effects in Drosophila result from chromosome rearrangements where normal genes, having been placed next to heterochromatin, are inactivated in some cells but not in others, thereby producing a variegated tissue. We have determined that the euchromatic breakpoints for three variegating white mutants are clustered and lie approximately 25 kb downstream of the white structural gene. In each case the white locus is adjoined in the heterochromatin to a mobile genetic element. Satellite sequences are not involved. We also demonstrate that revertants of the variegating mutant, wm4, are reinversions that leave the initial wm4-heterochromatic junction intact so that some heterochromatin-derived sequences remain joined to white at its new location. These results suggest a simple model for understanding the structure of heterochromatic domains and how variegating position effects may arise.