Pamela K. Geyer

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.

The role of insulator elements in defining domains of gene expression

Insulators are naturally occurring DNA sequences that protect transgenes from genomic position effects, thereby establishing independent functional domains within the chromosome. Recent studies have focused on the identification of the cis and trans requirements for insulator activity. These experiments demonstrate that insulators contain multiple components that cooperate to confer their unique properties. Additionally, they suggest that the mechanism of insulation is related to that of enhancer function. Two models of insulator can be considered: a domain boundary and a transcriptional decoy model.

On the molecular mechanism of gypsy-induced mutations at the yellow locus of Drosophila melanogaster.

We determined the nucleotide sequence of genomic DNA corresponding to the yellow gene. The limits of the transcribed region were deduced from sequence analysis of yellow larval and pupal cDNA clones. The yellow transcription unit is simple, composed of two exons which are processed identically in both developmental stages into a mRNA of 1990 bp. The predicted yellow protein has a mol. wt of 60,752 daltons and appears to be a secreted protein having a structural function and not an enzymatic role in pigmentation. We also characterized the spontaneous mutation y2 and a revertant of this allele to investigate the mutagenic effect of the gypsy element inserted into this locus. Our results show that this transposon is inserted at ‐700 bp and that the y2+ revertant resulted from excision of the gypsy element leaving behind a complete long terminal repeat (LTR). We conclude, therefore, that the gypsy element is neither inserted into a pupal specific intron or regulatory sequence supporting the hypothesis that mutagenesis is a result of transcriptional interference by the gypsy element on the yellow gene.

Genomic insulators: connecting properties to mechanism

Insulators are regulatory elements that establish independent domains of transcriptional activity within eukaryotic genomes. Insulators possess two properties: an anti-enhancer activity that blocks enhancer-promoter communication, and an anti-silencer activity that prevents the spread of repressive chromatin. Some insulators are composite elements with separable activities, while others employ a single mechanism to confer both properties. Recent studies focus on elucidating the molecular mechanisms of insulator function. Emerging themes support connections between insulators, transcriptional activators and topological chromosomal domains. Understanding these processes will provide insights into prevention of inappropriate regulatory interactions, knowledge that can be applied to gene therapies.

Tissue-specific transcriptional enhancers may act in trans on the gene located in the homologous chromosome: the molecular basis of transvection in Drosophila.

The y2 mutation resulted from the insertion of the gypsy element into the X‐linked yellow locus of Drosophila melanogaster. As a consequence of this insertion, transcriptional enhancers that control the expression of the yellow gene in the wings and body cuticle of adult flies are unable to act on the yellow promoter, resulting in a tissue‐specific phenotype characterized by mutant coloration in these structures. Some yellow null alleles (yn) are able to complement the y2 phenotype giving rise to near wild type y2/yn females. The molecular structure of the yellow locus in complementing and noncomplementing mutations was determined by cloning and sequencing the various alleles examined. From the information obtained in these studies, we propose a model suggesting that the complementing wild type phenotype of y2/yn flies might be due to the ability of functional wing and body cuticle transcriptional enhancers located in the yn locus to act in trans on the promoter of the yellow gene found in the y2‐containing chromosome. Furthermore, this transactivation is abolished by the presence of an intact promoter in cis, suggesting that promoter competition between the yellow genes located on each homolog precludes the activation in trans by transcriptional enhancers in favour of cis effects on their own promoter.

Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes.

The suppressor of Hairy‐wing [su(Hw)] protein mediates the mutagenic effects of the gypsy retrotransposon by blocking enhancer activity. These repressive effects are general, can occur over long distances and require that the su(Hw) protein is bound between the affected enhancer and promoter. The effects of the su(Hw) binding region on yolk protein (yp) gene expression were determined. These genes are regulated by shared enhancers in the intergenic region, which provided a method to examine whether an enhancer blocked by the su(Hw) protein remained functional. We demonstrate that a blocked enhancer is completely active, supporting the proposal that the su(Hw) protein is an insulator protein that acts by forming a new boundary in a pre‐existing chromatin domain, thereby preventing the interaction of regulatory elements located upstream of the insertion site with the promoter. In addition, we found that yp promoter function is not diminished by sharing enhancers, suggesting that these enhancers are not rate limiting for transcriptional activation. Lastly, our data indicate that yp promoter activity is interdependent, such that transcription from one promoter influences the level of activity of the linked promoter.

Regulation of Ribosomal Protein mRNA Content and Translation in Growth-Stimulated Mouse Fibroblasts

When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.

An endogenous Suppressor of Hairy-wing insulator separates regulatory domains in Drosophila

Insulators define independent domains of gene function throughout the genome. The Drosophila gypsy insulator was isolated from the gypsy retrotransposon as a region that contains a cluster of binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. To study the effects of the gypsy insulator on gene expression within a single genomic domain, targeted gene replacement was used to exchange the endogenous yellow gene, located at cytological location 1A, with a set of gypsy-modified yellow genes. Replaced yellow genes carried a gypsy insulator positioned between the yellow promoter and either the upstream or the downstream tissue-specific enhancers. Whereas the gypsy insulator blocked the function of the upstream enhancers at the endogenous location, the downstream enhancers were not blocked. Investigation of the 1A region revealed two clustered Su(Hw)-binding sites downstream of the yellow gene, named 1A-2, that bind Su(Hw) in vivo and possess enhancer blocking function. We propose that interaction between 1A-2 and the gypsy insulator permits activation of yellow expression by enhancers in the neighboring achaete-scute complex, causing an apparent absence of the block of the downstream yellow enhancers. Based on these data, we suggest that 1A-2 is an endogenous Su(Hw) insulator that separates regulatory domains within the Drosophila genome.

The role of Drosophila Lamin C in muscle function and gene expression

The inner side of the nuclear envelope (NE) is lined with lamins, a meshwork of intermediate filaments that provides structural support for the nucleus and plays roles in many nuclear processes. Lamins, classified as A- or B-types on the basis of biochemical properties, have a conserved globular head, central rod and C-terminal domain that includes an Ig-fold structural motif. In humans, mutations in A-type lamins give rise to diseases that exhibit tissue-specific defects, such as Emery-Dreifuss muscular dystrophy. Drosophila is being used as a model to determine tissue-specific functions of A-type lamins in development, with implications for understanding human disease mechanisms. The GAL4-UAS system was used to express wild-type and mutant forms of Lamin C (the presumed Drosophila A-type lamin), in an otherwise wild-type background. Larval muscle-specific expression of wild type Drosophila Lamin C caused no overt phenotype. By contrast, larval muscle-specific expression of a truncated form of Lamin C lacking the N-terminal head (Lamin C ΔN) caused muscle defects and semi-lethality, with adult ‘escapers’ possessing malformed legs. The leg defects were due to a lack of larval muscle function and alterations in hormone-regulated gene expression. The consequences of Lamin C association at a gene were tested directly by targeting a Lamin C DNA-binding domain fusion protein upstream of a reporter gene. Association of Lamin C correlated with localization of the reporter gene at the nuclear periphery and gene repression. These data demonstrate connections among the Drosophila A-type lamin, hormone-induced gene expression and muscle function.

A test of insulator interactions in Drosophila

Insulators are a class of elements that define independent domains of gene function. The Drosophila gypsy insulator is proposed to establish regulatory isolation by forming loop domains that constrain interactions between transcriptional control elements. This supposition is based upon the observation that insertion of a single gypsy insulator between an enhancer and promoter blocks enhancer function, while insertion of two gypsy insulators promotes enhancer bypass and activation of transcription. To investigate this model, we determined whether non‐gypsy insulators interacted with each other and with the gypsy insulator. Pairs of scs or scs′ insulators blocked enhancer function. Further, an intervening scs insulator did not block gypsy insulator interactions. Taken together, these data suggest that not all Drosophila insulators interact, with this property restricted to some insulators, such as gypsy. Three gypsy insulators inserted between an enhancer and promoter blocked enhancer function, indicating that gypsy insulator interactions may be restricted to pairs. Our studies imply that formation of loop domains may represent one of many mechanisms used by insulators to impart regulatory isolation.