Distant liaisons: long-range enhancer-promoter interactions in Drosophila.

Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancer sequences located several kilobases from the promoter. Existing models for how activator proteins function do not adequately explain long-range activation. Recent experiments in Drosophila on insulators that block enhancer-promoter interactions, interchromosomal activation, and mutants deficient in long-range activation are consistent with models in which facilitator factors that function between enhancers and promoters bring them into physical proximity of each other.

Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster.

The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. We found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. We propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.

Insulation of enhancer-promoter communication by a gypsy transposon insert in the Drosophila cut gene: cooperation between suppressor of hairy-wing and modifier of mdg4 proteins.

The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein.

The DNA-binding and enhancer-blocking domains of the Drosophila suppressor of Hairy-wing protein.

Mutations in the suppressor of Hairy-wing [su(Hw)] gene of Drosophila melanogaster can cause female sterility and suppress mutations that are insertions of the gypsy retrotransposon. Gypsy binds the protein (SUHW) encoded by su(Hw), and SUHW prevents enhancers promoter-distal to gypsy from activating gene transcription. SUHW contains 12 zinc fingers flanked by acidic N- and C-terminal domains. We examined the roles of each of the 12 zinc fingers in binding gypsy DNA and classified them into four groups: essential (fingers 6 through 10); beneficial but nonessential (fingers 1, 2, 3, and 11); unimportant (fingers 5 and 12); and inhibitory (finger 4). Because finger 10 is not required for female fertility but is essential for binding gypsy, these results imply that the SUHW-binding sites required for oogenesis differ in sequence from the gypsy-binding sites. We also examined the functions of the N- and C-terminal domains of SUHW by determining the ability of various deletion mutants to support female fertility and to alter expression of gypsy insertion alleles of the yellow, cut, forked, and Ultrabithorax genes. No individual segment of the N- and C-terminal domains of SUHW is absolutely required to alter expression of gypsy insertion alleles. However, the most important domain lies between residues 737 and 880 in the C-terminal domain. This region also contains the residues required for female fertility, and the fertility domain may be congruent with the enhancer-blocking domain. These results imply that SUHW blocks different enhancers and supports oogenesis by the same or closely related molecular mechanisms.

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.

Linear simian virus 40 DNA fragments exhibit a propensity for rolling-circle replication.

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.

Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila.

When the gypsy retrotransposon of Drosophila inserts between an enhancer and promoter it prevents the enhancer from activating transcription. Enhancers are blocked because the protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy. For example, gypsy insertions in an 85 kilobase region between a wing margin-specific enhancer and the promoter in the cut gene cause a cut wing phenotype that is suppressed by su(Hw) mutations. A temperature-sensitive combination of mutant su(Hw) alleles was used to investigate the mechanism by which SUHW blocks the cut wing margin enhancer. By shifting from the nonpermissive to the permissive temperature and vice versa at various stages in development it was found that active SUHW is only required around pupariation when the wing margin enhancer is active to cause a cut wing phenotype. This was true whether gypsy was in the embryonic control region near the promoter, or in the late larval control region near the wing margin enhancer. These results indicate that SUHW must be active only when an enhancer is active to block the enhancer. Furthermore, the observations also indicate that enhancer-blocking by SUHW is reversible and that it occurs soon after binding of active SUHW to gypsy DNA. These results are consistent with models in which SUHW structurally interferes with enhancer-promoter interactions.

The Drosophila melanogaster suppressor of Hairy-wing zinc finger protein has minimal effects on gene expression in Saccharomyces cerevisiae.

Many mutations in Drosophila melanogaster are gypsy retrotransposon insertions. Gypsy binds the protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene, and SUHW alters expression of surrounding genes. When gypsy is between an enhancer and promoter, SUHW blocks activation of transcription by the enhancer. Additionally, when gypsy is downstream of a promoter in a parallel orientation, SUHW increases truncation of transcripts at the poly(A) site in the gypsy 5' long terminal repeat, thereby decreasing the gene transcript levels. The effects of SUHW appear to involve fundamental and general mechanisms controlling gene expression because SUHW potentiates other poly(A) sites and blocks several enhancers in Drosophila. To investigate these mechanisms, SUHW was expressed in Saccharomyces cerevisiae. Although SUHW enters the nucleus and binds DNA in yeast, it has surprisingly minor effects on utilization of the CYC1 poly(A) site and transcription activation by a GAL upstream activation sequence. These observations indicate that the observed effects of SUHW on gene expression in Drosophila require specific interactions with other factors that are absent or unrecognizable in yeast.

Genes regulating the remote wing margin enhancer in the Drosophila cut locus.

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the suppressor of Hairy-wing protein (SUHW) of Drosophila. SUHW binds a DNA sequence in the gypsy retrotransposon and prevents enhancers promoter-distal to a gypsy insertion in a gene from activating without affecting promoter-proximal enhancers. Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions between scalloped, mastermind and Chip mutations indicate that mastermind and Chip act synergistically with scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of SUHW enhancer-blocking activity.

A proline-rich region in the Zeste protein essential for transvection and white repression by Zeste.

The DNA-binding protein encoded by the zeste gene of Drosophila activates transcription and mediates interchromosomal interactions such as transvection. The mutant protein encoded by the zeste1 (z1) allele retains the ability to support transvection, but represses white. Similar to transvection, repression requires Zeste-Zeste protein interactions and a second copy of white, either on the homologous chromosome or adjacent on the same chromosome. We characterized two pseudorevertants of z1 (z1-35 and z1-42) and another zeste mutation (z78c) that represses white. The z1 lesion alters a lysine residue located between the N-terminal DNA-binding domain and the C-terminal hydrophobic repeats involved in Zeste self-interactions. The z78c mutation alters a histidine near the site of the z1 lesion. Both z1 pseudorevertants retain the z1 lesion and alter different prolines in a proline-rich region located between the z1 lesion and the self-interaction domain. The pseudorevertants retain the ability to self-interact, but fail to repress white or support transvection at Ultrabithorax. To account for these observations and evidence indicating that Zeste affects gene expression through Polycomb group (Pc-G) protein complexes that epigenetically maintain chromatin states, we suggest that the regions affected by the z1, z78c, and pseudorevertant lesions mediate interactions between Zeste and the maintenance complexes.

Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes.

How enhancers are able to activate promoters located several kilobases away is unknown. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Here we show that Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation by a wing margin enhancer partly impaired by a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair.

Structure and expression of wild-type and suppressible alleles of the Drosophila purple gene.

Viable mutant alleles of purple (pr), such as prbw, exhibit mutant eye colors. This reflects low 6-pyruvoyl tetrahydropterin (PTP) synthase activity required for pigment synthesis. PTP synthase is also required for synthesis of the enzyme cofactor biopterin; presumably this is why some pr alleles are lethal. The prbw eye color phenotype is suppressed by suppressor of sable [su(s)] mutations. The pr gene was cloned to explore the mechanism of this suppression. pr produces two PTP synthase mRNAs: one constitutively from a distal promoter and one in late pupae and young adult heads from a proximal promoter. The latter presumably supports eye pigment synthesis. The prbw allele has a 412 retrotransposon in an intron spliced from both mRNAs. However, the head-specific mRNA is reduced > 10-fold in prbw and is restored by a su(s) mutation, while the constitutive transcript is barely affected. The Su(s) protein probably alters processing of RNA containing 412. Because the intron containing 412 is the first in the head-specific mRNA and the second in the constitutive mRNA, binding of splicing machinery to nascent transcripts before the 412 insertion is transcribed may preclude the effects of Su(s) protein.

A monocyte-derived factor interferes with detection of reverse transcriptase in HIV-1 infection.

Culture supernatants from the rabbit macrophage cell line 6083 infected with a retrovirus, human immunodeficiency virus type 1 (HIV-1), were negative for reverse transcriptase (RT) expression although the line was shown to be productively infected by all other criteria tested. Supernatants from uninfected cultures of 6083, the human monocyte line U937, and from freshly isolated peripheral human monocytes, were found to contain a monocyte-derived inhibitory factor (MDIF) which interferes with a standard assay for RT. MDIF is a heat-labile activity of approximately of 40 kD. Both substrates and products of the reverse transcriptase assay are degraded by MDIF which is not affected by reduction and alkylation of disulfide bonds. MDIF is inhibited by the addition of a particular thioated oligonucleotide (S-dG30) to the reaction mixture but this addition also inhibits RT. The optimum method to minimize MDIF interference in the RT assay is by addition of ethylene glycol bis-(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA); MDIF requires divalent cations for activity and has a strong preference for calcium which is preferentially chelated by EGTA. The potential presence of this inhibitory activity should be considered when using RT levels as a measure of retroviral infection.

Tooth loss in maintenance patients in a private periodontal practice.

A group of 162 maintenance patients, previously studied for compliance to suggested maintenance schedules, were surveyed for tooth loss over a 5-year period. The group was divided into those who complied to suggested maintenance schedules and those whose compliance was erratic. It was found that none of the patients who had complied to suggested maintenance schedules lost any teeth. In the erratic group, where all tooth loss occurred, it was found that the more often a patient presented for maintenance, the less likely he was to lose teeth. These findings are discussed in relation to current studies on efficacy of various therapies for periodontal diseases.