Heterochromatic self-association, a determinant of nuclear organization, does not require sequence homology in Drosophila

Chromosomes of higher eukaryotes contain blocks of heterochromatin that can associate with each other in the interphase nucleus. A well-studied example of heterochromatic interaction is the brown(Dominant) (bwD) chromosome of D. melanogaster, which contains an approximately 1.6-Mbp insertion of AAGAG repeats near the distal tip of chromosome 2. This insertion causes association of the tip with the centric heterochromatin of chromosome 2 (2h), which contains megabases of AAGAG repeats. Here we describe an example, other than bwD, in which distally translocated heterochromatin associates with centric heterochromatin. Additionally, we show that when a translocation places bwD on a different chromosome, bwD tends to associate with the centric heterochromatin of this chromosome, even when the chromosome contains a small fraction of the sequence homology present elsewhere. To further test the importance of sequence homology in these interactions, we used interspecific mating to introgress the bwD allele from D. melanogaster into D. simulans, which lacks the AAGAG on the autosomes. We find that D. simulans bwD associates with 2h, which lacks the AAGAG sequence, while it does not associate with the AAGAG containing X chromosome heterochromatin. Our results show that intranuclear association of separate heterochromatic blocks does not require that they contain the same sequence.     

Heterochromatic Trans-Inactivation of Drosophila White Transgenes

Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene, is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.     

Differential gene silencing by trans-heterochromatin in Drosophila melanogaster.

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin leading to the trans-inactivation of the wild-type allele in bw(D)/bw(+) heterozygous flies. This silencing is correlated with the localization of bw(+) to a region of the interphase nucleus containing centric heterochromatin. We have used a series of transgene constructs inserted in the vicinity of the bw locus to demarcate both the extent of bw(D) influence along the chromosome and the relative sensitivities of various genes. Examples of regulatory regions that are highly sensitive, moderately sensitive, and insensitive were found. Additionally, by using the same transgene at increasing distances from the bw(D) insertion site in trans we were able to determine the range of influence of the heterochromatic neighborhood in terms of chromosomal distance. When the transgene was farther away from bw, there was, indeed, a tendency for it to be less trans-inactivated. However, insertion site also influenced silencing: a gene 86 kb away was trans-inactivated, while the same transgene 45 kb away was not. Thus location, distance, and gene-specific differences all influence susceptibility to trans-silencing near a heterochromatic neighborhood. These results have important implications for the ability of nuclear positioning to influence the expression of large blocks of a chromosome.     

Analysis of chromatin structure of genes silenced by heterochromatin in trans

While heterochromatic gene silencing in cis is often accompanied by nucleosomal compaction, characteristic histone modifications, and recruitment of heterochromatin proteins, little is known concerning genes silenced by heterochromatin in trans. An insertion of heterochromatic satellite DNA in the euchromatic brown (bw) gene of Drosophila melanogaster results in bwDominant (bwD), which can inactivate loci on the homolog by relocation near the centric heterochromatin (trans-inactivation). Nucleosomal compaction was found to accompany trans-inactivation, but stereotypical heterochromatic histone modifications were mostly absent on silenced reporter genes. HP1 was enriched on trans-inactivated reporter constructs and this enrichment was more pronounced on adult chromatin than on larval chromatin. Interestingly, this HP1 enrichment in trans was unaccompanied by an increase in the 2MeH3K9 mark, which is generally thought to be the docking site for HP1 in heterochromatin. However, a substantial increase in the 2MeH3K9 mark was found on or near the bwD satellite insertion in cis, but did not spread further. These observations suggest that the interaction of HP1 with chromatin in cis is fundamentally different from that in trans. Our molecular data agree well with the differential phenotypic effect on bwD trans-inactivation of various genes known to be involved in histone modification and cis gene silencing.     

Analysis of copia sequence variation within and between Drosophila species

The sequences of the 5' long-terminal repeat (LTR) and adjacent leader regions of 27 full-length copia elements isolated from natural populations of Drosophila melanogaster, D. simulans, and D. mauritiana are presented. Phylogenetic analyses indicate that although D. melanogaster copia elements are distinct from those of D. simulans and D. mauritiana, the elements of these latter two species are not distinguishable from one another. LTRs and adjacent 5' leader regions of elements isolated from D. simulans and D. mauritiana are structurally similar to one another and carry substantial deletional variation mapping to regions previously identified as being of potential importance for copia expression.      

Interplay of developmentally regulated gene expression and heterochromatic silencing in trans in Drosophila

The brown(Dominant) (bw(D)) allele of Drosophila contains a heterochromatic block that causes the locus to interact with centric heterochromatin. This association silences bw(+) in heterozygotes (trans-inactivation) and is dependent on nuclear organizational changes later in development, suggesting that trans-inactivation may not be possible until later in development. To study this, a P element containing an upstream activating sequence (UAS)-GFP reporter was inserted 5 kb from the bw(D) insertion site. Seven different GAL4 driver lines were used and GFP fluorescence was compared in the presence or the absence of bw(D). We measured silencing in different tissues and stages of development and found variable silencing of GFP expression driven by the same driver. When UAS-GFP was not expressed until differentiation in the eye imaginal disc it was more easily trans-inactivated than when it was expressed earlier in undifferentiated cells. In contrast to some studies by other workers on silencing in cis, we did not find consistent correlation of silencing with level of expression or evidence of relaxation of silencing with terminal differentiation. We suggest that such contrasting results may be attributed to a potentially different role played by nuclear organization in cis and trans position-effect variegation.     

Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brown^Dominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.     

Sequence elements in cis influence heterochromatic silencing in trans

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin, which causes the locus to aberrantly associate with heterochromatin in interphase nuclei and silences the wild-type allele in heterozygotes. Transgenes placed near the bw(+) locus, in trans to bw(D), can also be silenced. The strength of silencing (called trans inactivation) varies with the regulatory sequences of the transgene and its distance away from the bw(D) insertion site in trans. In this study, we examine endogenous sequences in cis that influence susceptibility of a reporter gene to trans inactivation. Flanking deletions were induced in two parental lines containing P-element transgenes showing trans inactivation of the mini-white reporter. These new lines, which have mini-white under the influence of different endogenous sequence elements, now show varied ability to be silenced by bw(D). Determination of the deleted regions and the levels of mini-white expression and trans inactivation has allowed us to explore the correlation between cis sequence elements and susceptibility to trans inactivation and to identify a 301-bp sequence that acts as an enhancer of trans inactivation. Intriguingly, this region encompasses the upstream regions of two divergently transcribed genes and contains a sequence motif that may bind BEAF, a protein involved in delimiting chromatin boundaries.