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.     

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.     

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.     

Pairing-dependent mislocalization of a Drosophila brown gene reporter to a heterochromatic environment.

We describe the precise positioning of a reporter gene within heterochromatin where it may be silenced. A transposition of the 59E-60A region into pericentric heterochromatin ensnares distal 59E-60A via somatic pairing. The frequency with which a brown (bw) reporter gene in 59E is silenced is influenced by chromosomal configurations. Silencing occurs only when the bw+ reporter is unpaired due to heterozygosity with a deficiency, where the frequency of bw+ reporter expression is correlated with the extent of bw gene and flanking sequence present. Surprisingly, the frequency of pairing between the transposition in heterochromatin and distal 59E observed cytologically is indistinguishable from the frequency of pairing of homologous chromosomes at 59E in wild-type larval brains, regardless of configuration. Therefore, bringing a susceptible reporter gene into close proximity with heterochromatin does not necessarily affect its expression, but local pairing changes resulting from altered chromosomal configurations can lead to silencing. We also find that an ensnared distal copy of bw that is interrupted by a heterochromatic insertion enhances silencing. This demonstrates that bw can be simultaneously acted upon by pericentric and distal blocks of heterochromatin.     

Inactivation of reporter genes by cloned heterochromatic repeats of Drosophila melanogaster is accompanied by chromatin compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter gene mini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of the mini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-white have been found.     

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.     

Inactivation of Reporter Genes by Cloned Heterochromatic Repeats of Drosophila melanogaster is Accompanied by Chromatin Compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter genemini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of themini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-whitehave been found.     

The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects.

Mutations in the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of a number of tissue-specific mutations caused by insertion of a gypsy retrotransposon. The su(Hw) gene encodes a zinc finger protein which binds to a 430 bp region of gypsy shown to be both necessary and sufficient for its mutagenic effects. su(Hw) protein causes mutations by inactivation of enhancer elements only when a su(Hw) binding region is located between these regulatory sequences and a promoter. To understand the molecular basis of enhancer inactivation, we tested the effects of su(Hw) protein on expression of the mini-white gene. We find that su(Hw) protein stabilizes mini-white gene expression from chromosomal position-effects in euchromatic locations by inactivating negative and positive regulatory elements present in flanking DNA. Furthermore, the su(Hw) protein partially protects transposon insertions from the negative effects of heterochromatin. To explain our current results, we propose that su(Hw) protein alters the organization of chromatin by creating a new boundary in a pre-existing domain of higher order chromatin structure. This separates enhancers and silencers distal to the su(Hw) binding region into an independent unit of gene activity, thereby causing their inactivation.     

A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.     

Distance and Pairing Effects on the Brown(dominant) Heterochromatic Element in Drosophila

We examined the behavior of the brown(Dominant) (bw(D)) heterochromatic insertion moved to different locations relative to centric heterochromatin. Effects were measured as the degree of silencing of a wild-type brown eye pigment gene by bw(D) across a tandem duplication. A series of X-ray-induced effects were recovered at high frequency. Cis-acting enhancers were obtained by relocation of the duplication closer to autosomal heterochromatin. Enhancers were also recovered on the homologous chromosome when it was similarly rearranged, revealing a novel interhomologue effect whereby interactions occur between genetic elements near opposite ends of a chromosome arm rather than between paired alleles. Cis-acting suppressors were obtained as secondary rearrangements in which the duplication was moved farther away from heterochromatin. Suppression was correlated with loss of cytological association between bw(D) and the polytene chromocenter. Surprisingly, the distance from bw(D) to the chromocenter was not correlated with the strength of enhancement or suppression. We propose that bw(D) fails to coalesce with the chromocenter when its position along the chromosome places it beyond a threshold distance from heterochromatin, and this threshold depends upon the configuration of both the chromosome carrying bw(D) and its paired homologue.     

Characterization of the translation-dependent step during iron-regulated decay of transferrin receptor mRNA.

Iron regulates the stability of the mRNA encoding the transferrin receptor (TfR). When iron is scarce, iron regulatory proteins (IRPs) stabilize TfR mRNA by binding to the 3'-untranslated region. High levels of iron induce degradation of TfR mRNA; the translation inhibitor cycloheximide prevents this. To distinguish between cotranslational mRNA decay and a trans effect of translation inhibitors, we designed a reporter system exploiting the properties of the selectable marker gene thymidine kinase (TK). The 3'-untranslated region of human transferrin receptor, which contains all elements necessary for iron-dependent regulation of mRNA stability, was fused to the TK cDNA. In stably transfected mouse fibroblasts, the expression of the reporter gene was perfectly regulated by iron. Introduction of stop codons in the TK coding sequence or insertion of stable stem-loop structures in the leader sequence did not affect on the iron-dependent regulation of the reporter mRNA. This implies that global translation inhibitors stabilize TfR mRNA in trans. Cycloheximide prevented the destabilization of TfR mRNA only in the presence of active IRPs. Inhibition of IRP inactivation by cycloheximide or by the specific proteasome inhibitor MG132 correlated with the stabilization of TfR mRNA. These observations suggest that inhibition of translation by cycloheximide interferes with the rate-limiting step of iron-induced TfR mRNA decay in a trans-acting mechanism by blocking IRP inactivation.     

The controlling element Ds at the Shrunken locus in Zea mays: structure of the unstable sh-m5933 allele and several revertants

We have analyzed the structure of the Shrunken (Sh) locus in a strain containing an unstable recessive mutation, sh-m5933, caused by the transposable controlling element Dissociation (Ds). We have also analyzed nine spontaneous Sh revertant alleles. The sh-m5933 allele contains a 30 kb insertion at the Sh locus, as well as a duplication that includes part of the insertion and the Sh locus sequence on the 5' side of the insertion site. The revertants continue to show Ds-mediated chromosome breakage at the Sh locus, have an intact Sh locus from which the insertion has been excised, and retain the duplication. One of the nine revertant alleles has a 2 kb deletion at the junction between the Sh locus and the insertion sequence in the duplicated segment of the locus. The revertant also shows a temporal change in the pattern of somatic chromosome breakage, implicating the junction sequence as the site of Ds-mediated chromosome breakage.     

Mutation of Drosophila melanogaster glass-like suppress expression of mini-white transgenes and dependence on their genomic environment

Pleiotropic recessive mutation glass-like (gl-l) found in region 8C-10E of the X chromosome was shown to cause glass-like eyes having no boundaries between facets and a nonuniform pigment distribution in the presence of the endogenous white gene. The gl-l mutation completely inhibited expression of the mini-white transgene contained in several constructs, but the effect depended on the site of construct integration in the genome. The mutation had no effect on the expression of the white transgene having the enhancer and flanked by insulators. The gl-l mutation did not affect the extent of mosaic eye pigmentation when a construct with mini-white was inserted in the telomeric or pericentric region. However, in most cases it completely inhibited the mosaic mini-white expression when cloned heterochromatic repeats were adjacent to the reporter gene in a construct. The gl-l gene was assumed to play a role in the formation of the chromatin structure, because the effect of its mutation on expression of the white transgene depended on the transgene insertion site, the presence of insulators or an enhancer in the vicinity of the transgene, and on the adjacent heterochromatic repeats.     

Telomeric associated sequences of Drosophila recruit polycomb-group proteins in vivo and can induce pairing-sensitive repression

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. Here, we present the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes revealed a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE).