Biomedical subjects

P Schedl

Publications and source records attributed to P Schedl.

At least 19 recordsLinked to original sources

PubMed1996

Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex.

Fab-7 deletions in the bithorax complex have a novel gain-of-function phenotype, typically transforming parasegment 11 (PS11) into PS12 identity. Genetic analysis indicates that removal of the Fab-7 element results in the fusion of the iab-6 (PS11) and iab-7 (PS12) cis-regulatory domains into a single regulatory domain that inappropriately regulates Abdominal-B in PS11. This has led to the hypothesis that Fab-7 is a chromatin domain boundary that normally functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains. We use several different enhancer blocking assays to demonstrate that Fab-7 has the insulating properties expected of a domain boundary. We define a minimal fragment of Fab-7 sufficient for enhancer blocking, and demonstrate that it is completely distinct from an adjacent Polycomb-dependent silencer. We compare Fab-7 to the su(Hw) insulator element, and show that Fab-7 enhancer blocking activity is intermediate between that of five and twelve reiterated binding sites for the Su(Hw) protein. These results support the model that Fab-7 functions as a domain boundary within the context of the bithorax complex, making Fab-7 one of the first boundary elements that is known to have an essential function in vivo.

Animals↗

PubMed1996

Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila.

In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination -- when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of the length except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently incapable of altering the splicing pattern of these pre-mRNAs.

Alternative Splicing↗

PubMed1996

The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division.

The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle.

Animals↗

PubMed1996

Splicing of the drosophila Sex-lethal early transcripts involves exon skipping that is independent of Sex-lethal protein.

mRNAs from the early Sex-lethal promoter, Sxl-Pe, encode embryonic Sxl proteins that function to activate the Sxl autoregulatory loop. They do so by directing the female-specific splicing of the first transcripts expressed from the late or maintenance promoter, Sxl-Pm. The early promoter is located, however, upstream not downstream of the translation terminating male-specific exon, L3, and upstream of the second Sxl-Pm exon, L2. If the Sxl proteins expressed from Sxl-Pe are to provide the mechanism for bypassing the normal requirement for Sxl protein in the female-specific splicing of transcripts from Sxl-Pm, then what is the mechanism for skipping L2 and L3 in the processing of transcripts from Sxl-Pe? In the studies reported here, we have generated a report construct to examine the splicing of Sxl-Pe transcripts. Our results indicate that neither specific maternal products, Sxl protein, nor an X chromosome to autosome ratio of 1 are required for the processing of the embryonic mRNAs. We also found that none of the three genes, snf, virilizer, and fl(2)d, which when mutated alter the female-specific processing of Sxl-Pm transcripts, alter the generation of the early splice. Skipping to intervening exons to generate an open reading frame that will encode the Sxl early proteins appears to be an intrinsic property of initiating the early Sxl RNAs within the first intron of the Sxl-Pm maintenance transcription unit.

Animals↗

PubMed1995

Selection and maintenance of sexual identity in the Drosophila germline.

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.

Animals↗

PubMed1995

Multiple response elements in the Sex-lethal early promoter ensure its female-specific expression pattern.

The choice of sexual identity in somatic tissues of the fruit fly Drosophila melanogaster is determined early in embryogenesis by the X-chromosome-to-autosome (X/A) ratio. The system that signals the X/A ratio selects the sexual development pathway by determining the activity state of the binary switch Sex-lethal (Sxl). In 2X/2A animals, the X/A signalling system turns the Sxl gene on, ultimately activating an RNA-splicing autoregulatory feedback loop which serves to maintain the female state during the remainder of development. In 1X/2A animals, this autoregulatory feedback loop is not activated and the male state is subsequently maintained by the default splicing machinery. In the studies reported here, we have examined how the X/A signalling system controls the initial choice of sexual identity through its action on a special early embryonic Sxl promoter, Sxl-Pe. We show that in the early embryo, the activity of Sxl-Pe is controlled in a highly dose-sensitive fashion by the genes on the X chromosome that function as numerator elements and by genes located on the autosomes that function as denominator elements. Functional dissection of Sxl-Pe indicates that activating the promoter in females requires the cumulative action of multiple numerator genes which appear to exert their effects through reiterated cis-acting target sites in the promoter. Conversely, maintaining the promoter silent in males requires the repressive activities of denominator genes, and at least one of the denominator genes also appears to function through target sequences within the promoter.

Animals↗

PubMed1995

The miti-mere and pdm1 genes collaborate during specification of the RP2/sib lineage in Drosophila neurogenesis.

We have investigated (i) the role of pdm1, a Drosophila POU gene, during the elaboration of the GMC-1-->RP2/sib lineage and (ii) the functional relationship between pdm1 and the closely linked second POU gene, miti-mere, in this lineage. We show that deletion of pdm1 causes a partially penetrant GMC-1 defect, while deletion of both miti and pdm1 results in a fully penetrant defect. This GMC-1 defect in miti- and pdm1- embryos can be rescued by the pdm1 or miti transgene. Rescue is observed only when these genes are expressed at the time of GMC-1 formation. Overexpression of pdm1 or miti well after GMC-1 is formed results in the duplication of RP2 and/or sib cells. Our results indicate that both genes are required for the normal development of this lineage and that the two collaborate during the specification of GMC-1 identity.

Animals↗

PubMed1995

scute (sis-b) function in Drosophila sex determination.

The primary sex determination signal, the X chromosome-to-autosome (X/A) ratio, controls the choice of sexual identity in the Drosophila melanogaster embryo by regulating the activity of the early promoter of the Sex-lethal gene, Sxl-Pe. This promoter is activated in females (2X/2A), while it remains off in males (1X/2A). Promoter activation in females is dependent upon X-linked numerator genes. One of these genes, sisterless-b (sis-b), corresponds to the scute (sc) locus of the achaete-scute complex, and it encodes a helix-loop-helix transcription factor. In the studies reported here we have used monoclonal antibodies to study the expression and functioning of the sc(sis-b) protein. Sc is first detected at nuclear cycle 6 to 7, well before Sxl-Pe is first active. At this stage, the protein is in the cytoplasm, not the nucleus. Only after the formation of the syncytial blastoderm, at nuclear cycle 10 to 11, does a substantial fraction of the protein enter the nucleus, and this nuclear import closely coincides with the initial activation of Sxl-Pe. Consistent with the idea that the dose of sc(sis-b) is critical for its function as an X-chromosome counting element, wild-type syncytial blastoderm embryos could be grouped into two classes based on the level of protein. Western blot (immunoblot) analysis demonstrates that this difference in protein level correlates directly with the activity state of the Sxl gene. Finally, we provide the first direct evidence that Sc forms heteromeric complexes in vivo in early embryos with the maternally derived helix-loop-helix protein Daughterless. This in vivo complex is likely to be critical for Sc function in Sxl-Pe activation.

Animals↗

PubMed1994

Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

The Drosophila 87A7 heat shock locus is bordered, on the proximal and distal sides, by two special chromatin structures, scs and scs'. Each structure is characterized by two sets of nuclease-hypersensitive sites, located within moderately G/C-rich DNA, flanking an A/T-rich nuclease-resistant region. scs and scs' have been shown to insulate a white reporter gene from position effects and to prevent enhancer-promoter interactions. These and other properties suggest scs and scs' might function as chromatin domain boundaries. To identify the DNA sequences which are essential for the insulating activity of scs we used an enhancer blocking assay based on the white gene. Sequences capable of suppressing activation of white by its upstream enhancer elements reside within a 900 bp DNA fragment corresponding to the scs chromatin structure. Within this region, DNA fragments associated with the two nuclease-hypersensitive regions are essential for full enhancer blocking activity, while the central A/T-rich region is dispensable. Deletions which remove part of the hypersensitive regions result in intermediate levels of white activity. Insulating activity can, however, be reconstituted by multimerizing DNA fragments from either hypersensitive region. Our results suggest that the scs boundary is assembled from a discrete number of functionally redundant DNA sequences located within both hypersensitive regions and that boundaries act by decreasing the frequency of enhancer-promoter interactions. We also show that certain types of position effects, like those involved in dosage compensation, are not efficiently blocked by scs.

ATP-Binding Cassette Transporters↗

PubMed1994

Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.

A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.

Animals↗

PubMed1994

The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity.

The orb gene of Drosophila encodes sex-specific germ-line proteins that contain two RRM-type RNA-binding domains. Here we report the distribution of Orb protein in wild-type, tumorous, and orb mutant ovaries. The wild-type distribution of Orb protein during oogenesis resembles that of its RNA, preferentially accumulating in the cytoplasm of the developing oocyte shortly after the formation of the 16-cell cyst. As anticipated from its germ-line expression, mutations in orb lead to female sterility. Analysis of the effect of orb mutants on the distribution of RNAs known to be required for oocyte differentiation and polarity suggests that orb functions in RNA localization at multiple points during oogenesis. In addition, phenotypic characterization of the orb mutants indicates that the gene is required early in oogenesis for formation of the 16-cell cyst. It then functions in the differentiation of the oocyte and is required for the three-dimensional reorganization of the germ cells in the cyst as well as for the establishment of normal germ-line-soma interactions in the egg chamber.

Animals↗

PubMed1994

Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis.

The targeting of positional information to specific regions of the oocyte or early embryo is one of the key processes in establishing anterior-posterior and dorsal-ventral polarity. In many developmental systems, this is accomplished by localization of mRNAs. The germ line-specific Drosophila orb gene plays a critical role in defining both axes of the developing oocyte, and its mRNA is localized in a complex pattern during oogenesis. We have identified a 280-bp sequence from the orb 3' untranslated region capable of reproducing this complex localization pattern. Furthermore, we have found that multiple cis-acting elements appear to be required for proper targeting of orb mRNA.

Animals↗

PubMed1994

RNA binding by Sxl proteins in vitro and in vivo.

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

Animals↗

PubMed1994

The Drosophila miti-mere gene, a member of the POU family, is required for the specification of the RP2/sibling lineage during neurogenesis.

The Drosophila POU gene miti-mere (previously known as pdm2) has a complex spatial and temporal pattern of expression during early development; initially it is expressed in gap-gene-like pattern, then in 14 stripes and finally in a subset of the cells in the developing CNS and PNS. To study the function of this gene during development, we generated a 'synthetic anti-morphic mutation' by expressing a truncated version of the miti protein from a constitutive hsp83 and an inducible hsp70 promoter. We show that these delta miti transgenes behave like classical antimorphic mutations. Using these dominant negative transgenes, together with deletions and a duplication for the gene, we show that miti is required during segmentation and neurogenesis. We have also used temperature-shift experiments with the hsp70 delta miti transgene to demonstrate that miti function in segmentation is distinct and separable from its function during neurogenesis. In segmentation, miti appears to be required in the specification of the segments A2 and A6. In the CNS, miti is required for the elaboration of the NB4-2-->GMC-1-->RP2/sib lineage. miti is initially required in this lineage to establish the identity of the parental ganglion mother cell, GMC-1. miti must then be down-regulated to allow the asymmetric division of GMC-1 into the RP2 and its sibling cell.

Animals↗

PubMed1993

The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex.

An extremely large cis-regulatory region generates the parasegment-specific expression patterns of the homeotic genes in the bithorax complex. We present evidence supporting the idea that this cis-regulatory region is subdivided into independent cis-regulatory domains. We describe a Ubx-lacZ transposon which is inserted into one of these domains, iab-7. The PS12-specific pattern of LacZ expression from this reporter indicates that it is subject to the control of the iab-7 cis-regulatory domain, but is protected from the effects of adjacent regulatory domains. Protection on the proximal side appears to be provided by the Fab-7 boundary element. Deletion of this boundary results in the ectopic activation of iab-7 in PS11 (where the iab-6 cis-regulatory domain normally functions). We show that the Fab-7 boundary, like other boundaries, has an unusual chromatin structure.

Animals↗

PubMed1993

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

Animals↗

PubMed1993

Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site.

The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.

Animals↗

PubMed1993

Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism.

In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of genes whose expression is regulated by alternative splicing [B. S. Baker, Nature (London) 340:521-524, 1989; J. Hodgkin, Cell 56:905-906, 1989]. The master switch gene in this hierarchy is Sex-lethal. Sex-lethal is turned on only in females, and an autoregulatory feedback loop which controls alternative splicing maintains this state (L. R. Bell, J. I. Horabin, P. Schedl, and T. W. Cline, Cell 65:229-239, 1991; L. N. Keyes, T. W. Cline, and P. Schedl, Cell 68:933-943, 1992). Sex-lethal also promotes female differentiation by controlling the splicing of RNA from the next gene in the hierarchy, transformer. Sosnowski et al. (B. A. Sosnowski, J. M. Belote, and M. McKeown, Cell 58:449-459, 1989) have shown that the mechanism for generating female transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. We have tested whether an activation or a blockage mechanism is involved in Sex-lethal autoregulation. The male exon of Sex-lethal with flanking splice sites was placed into the introns of heterologous genes. Our results support the blockage mechanism. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. This and the observation that the splice signals at the exon are suboptimal are discussed with regard to alternate splicing.

Alternative Splicing↗