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.     

Constitutive heterochromatin and transposable elements in Drosophila melanogaster

Several families of transposable elements (TEs), most of them belonging to the retrotransposon catagory, are particularly enriched in Drosophila melanogaster constitutive heterochromatin. The enrichment of TE-homologous sequences into heterochromatin is not a peculiar feature of the Drosophila genome, but appears to be widespread among higher eukaryotes. The constitutive heterochromatin of D. melanogaster contains several genetically active domains; this raises the possibility that TE-homologous sequences inserted into functional heterochromatin compartments may be expressed. In this review, I present available data on the genetic and molecular organization of D. melanogaster constitutive heterochromatin and its relationship with transposable elements. The implications of these findings on the possible impact of heterochromatic TEs on the function and evolution of the host genome are also discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transposable elements as artisans of the heterochromatic genome in Drosophila melanogaster

Over 50 years ago Barbara McClintock discovered that maize contains mobile genetic elements, but her findings were at first considered nothing more than anomalies. Today it is widely recognized that transposable elements have colonized all eukaryotic genomes and represent a major force driving evolution of organisms. Our contribution to this special issue deals with the theme of transposable element-host genome interactions. We bring together published and unpublished work to provide a picture of the contribution of transposable elements to the evolution of the heterochromatic genome in Drosophila melanogaster. In particular, we discuss data on 1) colonization of constitutive heterochromatin by transposable elements, 2) instability of constitutive heterochromatin induced by the I factor, and 3) evolution of constitutive heterochromatin and heterochromatic genes driven by transposable elements. Drawing attention to these topics may have direct implications on important aspects of genome organization and gene expression.     

The cAMP signaling pathway regulates Epe1 protein levels and heterochromatin assembly

The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1⁺ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.     

Partial inversion of the secondary constriction of chromosome 9. Does it exist?

Summary Pericentric inversion of chromosome 9, a common abnormality, has been much studied because of its possible genetic effect. Apart from total inversion, in which the whole heterochromatic segment of chromosome 9 appears to be situated on the short arm, some authors describe partial inversion, in which the heterochromatin is found partly on the long arm and partly on the short arm.Our study indicates that firstly, the heterochromatic segment of chromosome 9 is composed of two biochemically different subunits: the heterochromatin of the centromere itself and the heterochromatin of the secondary constriction. Secondly, it suggests that partial inversion of the secondary constriction of chromosome 9 is an unusual event, as the majority of published cases can be interpreted as the result of an increase in the centromeric heterochromatin without alteration of the secondary constriction.Supported by grants from INSERM (A.T.P. 79-110)     

Effect of C-banded heterochromatin on centromere separation

The present study is to determine the effects of centromeric heterochromatin on centromere separation. Amniotic cell cultures in which the centromeric heterochromatin of one chromosome was at least twice as large (qh+) as the heterochromatin (qh) in the homologous chromosome were selected. Fifteen amniotic cell samples with 1qh+, 9qh+ or 16qh+ were studied. The size of the centromeric heterochromatin was directly correlated with the delay in centromere separation. The chromosome with the smaller centromeric heterochromatin tended to show earlier centromere separation than the homologue with the larger heterochromatin. Our results suggest that the quantity of centromeric heterochromatin may influence the genetic control of centromere separation.     

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

Chromatin structure and physical mapping of chromosome 6 of potato and comparative analyses with tomato

Potato (Solanum tuberosum) has the densest genetic linkage map and one of the earliest established cytogenetic maps among all plant species. However, there has been limited effort to integrate these maps. Here, we report fluorescence in situ hybridization (FISH) mapping of 30 genetic marker-anchored bacterial artificial chromosome (BAC) clones on the pachytene chromosome 6 of potato. The FISH mapping results allowed us to define the genetic positions of the centromere and the pericentromeric heterochromatin and to relate chromatin structure to the distribution of recombination along the chromosome. A drastic reduction of recombination was associated with the pericentromeric heterochromatin that accounts for ~28% of the physical length of the pachytene chromosome. The pachytene chromosomes 6 of potato and tomato (S. lycopersicum) share a similar morphology. However, distinct differences of heterochromatin distribution were observed between the two chromosomes. FISH mapping of several potato BACs on tomato pachytene chromosome 6 revealed an overall colinearity between the two chromosomes. A chromosome inversion was observed in the euchromatic region of the short arms. These results show that the potato and tomato genomes contain more chromosomal rearrangements than those reported previously on the basis of comparative genetic linkage mapping.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.     

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.     

Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.     

Position Effect Variegation in Drosophila Melanogaster: Relationship between Suppression Effect and the Amount of Y Chromosome

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.     

Targeting heterochromatin formation to transposable elements in Drosophila: Potential roles of the piRNA system

Successful heterochromatin formation is critical for genome stability in eukaryotes, both to maintain structures needed for mitosis and meiosis and to silence potentially harmful transposable elements. Conversely, inappropriate heterochromatin assembly can lead to inappropriate silencing and other deleterious effects. Hence targeting heterochromatin assembly to appropriate regions of the genome is of utmost importance. Here we focus on heterochromatin assembly in Drosophila melanogaster, the model organism in which variegation, or cell-to-cell variable gene expression resulting from heterochromatin formation, was first described. In particular, we review the potential role of transposable elements as genetic determinants of the chromatin state and examine how small RNA pathways may participate in the process of targeted heterochromatin formation.     

Cytogenetic and molecular characterization of heterochromatin gene models in Drosophila melanogaster

In the past decade, genome-sequencing projects have yielded a great amount of information on DNA sequences in several organisms. The release of the Drosophila melanogaster heterochromatin sequence by the Drosophila Heterochromatin Genome Project (DHGP) has greatly facilitated studies of mapping, molecular organization, and function of genes located in pericentromeric heterochromatin. Surprisingly, genome annotation has predicted at least 450 heterochromatic gene models, a figure 10-fold above that defined by genetic analysis. To gain further insight into the locations and functions of D. melanogaster heterochromatic genes and genome organization, we have FISH mapped 41 gene models relative to the stained bands of mitotic chromosomes and the proximal divisions of polytene chromosomes. These genes are contained in eight large scaffolds, which together account for approximately 1.4 Mb of heterochromatic DNA sequence. Moreover, developmental Northern analysis showed that the expression of 15 heterochromatic gene models tested is similar to that of the vital heterochromatic gene Nipped-A, in that it is not limited to specific stages, but is present throughout all development, despite its location in a supposedly "silent" region of the genome. This result is consistent with the idea that genes resident in heterochromatin can encode essential functions.