The MCP silencer of the Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for the maintenance of repression.

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.

A silencer is required for maintenance of transcriptional repression throughout Drosophila development.

Transcriptional silencing by the Polycomb Group of genes maintains the position-specific repression of homeotic genes throughout Drosophila development. The Polycomb Group of genes characterized to date encode chromatin-associated proteins that have been suggested to form heterochromatin-like structures. By studying the expression of reporter genes, we have identified a 725 bp fragment, called MCP725, in the homeotic gene Abdominal-B, that accurately maintains position-specific silencing during proliferation of imaginal cells. Silencing by MCP725 requires the Polycomb and the Polycomblike genes, indicating that it contains a Polycomb response element To investigate the mechanisms of transcriptional silencing by MCP725, we have studied its temporal requirements by removing MCP725 from the transgene at various times during development. We have discovered that excision of MCP725 during larval stages leads to loss of silencing. Our findings indicate that the silencer is required for the maintenance of the repressed state throughout cell proliferation. They also suggest that propagation of the silenced state does not occur merely by templating of a heterochromatin structure by virtue of protein-protein interactions. Rather, they suggest that silencers play an active role in the maintenance of the position-specific repression throughout development.

Drosophila midgut morphogenesis requires the function of the segmentation gene odd-paired.

Development of the Drosophila embryonic midgut is dependent on a number of genes, including tinman and bagpipe, which are required for formation of the visceral mesoderm, and the homeotic genes and their targets, which act locally in the visceral mesoderm to direct formation of specific midgut constrictions. Here we report the identification and characterization of another gene, odd-paired (opa), required for normal midgut development. opa, first identified by its pair-rule mutant phenotype in the larval cuticle, encodes a protein containing putative DNA binding domains and other hallmarks of transcriptional regulators. We have positively identified the cloned gene as opa by mapping two null mutations to the open reading frame of the putative opa transcript; these mutations disrupt the open reading frame and generate predicted proteins lacking the DNA binding domain. We demonstrate that opa function is required for formation of the three characteristic midgut constrictions. To understand the mechanisms by which opa contributes to constriction formation, we have analyzed the expression and function of opa throughout midgut development. In the cellular blastoderm, opa is expressed ubiquitously in the ectoderm and mesoderm precursors throughout the presumptive segmented region. As development proceeds, opa expression ceases briefly both in the ectoderm and in the underlying mesodermal cells that later become the visceral mesoderm. We show that in opa mutants the visceral mesoderm is interrupted, evidently due to abnormal expression of bagpipe, a homeodomain gene required for the formation of the visceral mesoderm. At early stages of development in opa mutants, interruptions in the visceral mesoderm are observed at many positions along the anterior-posterior axis. As development proceeds, interruptions are less frequently observed; however, one interruption, coincident with the Antennapedia expression and variability of Ultrabithorax expression in opa mutants. From these observations, we infer that the loss of at least the first and second midgut constrictions in opa mutants is the result of defects first evident in the early stages of visceral mesoderm development.(ABSTRACT TRUNCATED AT 400 WORDS)

On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor.

The approximately 200 distinct neurons comprising each hemisegment of the Drosophila embryonic CNS are derived from a stereotypic array of approximately 30 progenitor stem cells, called neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce several smaller ganglion mother cells (GMCs), each of which, in turn, divides to produce two neurons and/or glia cells. To understand the process by which cell type diversity is generated in the CNS, we are focusing on identifying genes that affect cell identity in the NB4-2 lineage from which the RP2 motoneuron is derived. We show here that within the early part of the NB4-2 lineage, two closely linked and structurally related POU homeo domain genes, pdm-2 (dPOU28) and pdm-1 (dPOU19), both encode proteins that accumulate to high levels only in the first GMC (GMC4-2a) and not in its progeny, the RP2 motoneuron. Our results from the genetic and developmental analysis of pdm-1 and pdm-2 demonstrate that these genes are not required for the birth of GMC4-2a; however, they are both involved in specifying the identity of GMC4-2a and, ultimately, in the genesis of RP2 neurons, with pdm-2 being the more dominant player in this process. In mutant animals where both pdm-1 and pdm-2 functions are removed, GMC4-2a fails to express markers consistent with a GMC4-2a identity and no mature (Eve protein expressing) RP2 neurons are produced. We demonstrate that in some mutant combinations in which no mature RP2 neurons are produced, some GMC4-2a cells can nevertheless divide. Hence, the failure of the POU mutants to produce mature RP2 neurons is not attributable to a block in GMC4-2a cell division per se but, rather, because the GMC4-2a cells fail to acquire their correct cellular identity.

Cis and trans interactions between the iab regulatory regions and abdominal-A and abdominal-B in Drosophila melanogaster.

The infra-abdominal (iab) elements in the bithorax complex of Drosophila melanogaster regulate the transcription of the homeotic genes abdominal-A (abd-A) and Abdominal-B (Abd-B) in cis. Here we describe two unusual aspects of regulation by the iab elements, revealed by an analysis of an unexpected complementation between mutations in the Abd-B transcription unit and these regulatory regions. First, we find that iab-6 and iab-7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. In addition, we show that a transposed Abd-B transcription unit and promoter on the Y chromosome can be activated by iab elements located on the third chromosome. These results suggest that the iab regions can regulate their target promoter located at a distant site in the genome in a manner that is much less dependent on homologue pairing than other transvection effects. The iab regulatory regions may have a very strong affinity for the target promoter, allowing them to interact with each other despite the inhibitory effects of chromosomal rearrangements. Second, by generating abd-A mutations on rearrangement chromosomes that break in the iab-7 region, we show that these breaks induce the iab elements to switch their target promoter from Abd-B to abd-A. These two unusual aspects of iab regulation are related by the iab-7 breakpoint chromosomes that prevent iab elements from acting on Abd-B and allow them to act on abd-A. We propose that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter.

Cell-type-specific mechanisms of transcriptional repression by the homeotic gene products UBX and ABD-A in Drosophila embryos.

The homeotic genes of Drosophila melanogaster, which are required for specification of segmental identities, encode proteins capable of regulating gene expression. We have chosen to study the organization and function of a regulatory target in an attempt to learn how homeotic gene products provide appropriate transcriptional controls. We identified 30 common binding sites for the proteins encoded by the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes within a negatively regulated target, the P2 promoter of the Antennapedia (Antp) gene. By systematically mutagenizing binding sites and observing the resulting P2 expression pattern in embryos, we have found evidence for cell-type-specific interactions that are mediated by these sequences. In certain neuronal cells, UBX and ABD-A proteins appear to repress by competing for common binding sites with another homeodomain protein, which we propose to be ANTP acting to induce P2 transcription in an autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A repress by counteracting the function of a factor acting at independent sites. The latter mechanism of repression requires only that multiple homeodomain binding sequences be present and is not dependent on any particular binding site.

Ectopic expression of UBX and ABD-B proteins during Drosophila embryogenesis: competition, not a functional hierarchy, explains phenotypic suppression.

The Abdominal-B (Abd-B) gene, a member of the bithorax complex (BX-C), specifies the identities of parasegments (PS) 10-14 in Drosophila. Abd-B codes for two structurally related homeodomain proteins, ABD-B m and ABD-B r, that are expressed in PS10-13 and PS14-15, respectively. Although ABD-B m and r proteins have distinct developmental functions, ectopic expression of either protein during embryogenesis induces the development of filzkörper and associated spiracular hairs, structures normally located in PS13, at ectopic sites in the larval thorax and abdomen. These results suggest that other parasegmental differences contribute to the phenotype specified by ABD-B r activity in PS14. Both ABD-B m and r repress the expression of other homeotic genes, such as Ubx and abd-A, in PS10-14. However, the importance of these and other cross-regulatory interactions among homeotic genes has been questioned. Since ectopic UBX protein apparently failed to transform abdominal segments, González-Reyes et al. (González-Reyes, A., Urquía, N., Gehring, W.J., Struhl, G. and Morata, G. (1990). Nature 344, 78-80) proposed a functional hierarchy in which ABD-A and ABD-B activities override UBX activity. We tested this model by expressing UBX and ABD-B m proteins ectopically in wild-type and BX-C-deficient embryos. Ectopic ABD-B m does not prevent transformations induced by ectopic UBX. Instead, ectopic UBX and ABD-B m proteins compete for the specification of segmental identities in a dose-dependent fashion. Our results support a quantitative competition among the homeotic proteins rather than the existence of a strict functional hierarchy. Therefore, we suggest that cross-regulatory interactions are not irrelevant but are important for repressing the expression of competing homeotic proteins. To explain the apparent failure of ectopic UBX to transform the abdominal segments, we expressed UBX at different times during embryonic development. Our results show that ectopic UBX affects abdominal cuticular identities if expressed during early stages of embryogenesis. In later embryonic stages, abdominal segments become resistant to transformation by ectopic UBX while thoracic segments remain susceptible. Head segments also show a similar stage-dependent susceptibility to transformation by ectopic UBX in early embryogenesis but become resistant in later stages. These results suggest that abdominal and head identities are determined earlier than are thoracic identities.

Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster.

The abdominal-A (abd-A) gene, a member of the bithorax complex, is required for the correct identity of parasegments (PS) 7 through 13. Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adult flies. Here, we describe a developmental and molecular analysis of the role of iab-4 functions in gonadal development. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. To localize the sites that require iab-4 function, we have analyzed animals chimeric for the mutant and wild-type cells. These chimeras were generated by three kinds of transplantation experiments: pole cells, embryonic somatic nuclei or larval ovaries. Our results suggest that iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue.

Characterization of two Drosophila POU domain genes, related to oct-1 and oct-2, and the regulation of their expression patterns.

We have characterized two genes from Drosophila melanogaster that encode proteins with POU domains showing a high degree of identity with the human Oct-1 and Oct-2 transcription factors. These POU domain genes, pdm-1 and pdm-2, are expressed at high levels during early embryogenesis and at lower levels throughout the rest of development. Both genes are expressed as two stripes in the presumptive abdominal region during the blastoderm stage, followed by thirteen stripes in the germ band extended stage. This pattern of expression is altered in mutants for a gap gene (hunchback) and a pair-rule gene (fushi tarazu). In later stage embryos, both pdm-1 and pdm-2 are expressed in selected neuroblasts in the ventral nervous system, with higher levels in the three thoracic segments and lower levels in the abdominal segments. The low level of expression in the abdominal segments is maintained by the genes within the bithorax complex (BX-C). We have also identified the cells in the dorsal and lateral clusters of the peripheral nervous system that express pdm-1 and pdm-2, and show that some of these cells derive from lineages that require BX-C functions. Together, these results suggest that previously characterized members of the embryonic regulatory hierarchy specify the patterns of the POU domain gene expression, which, in turn, function during neurogenesis and perhaps in earlier stages.

Molecular definition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene.

The Abdominal-B (Abd-B) gene, a member of the Drosophila bithorax complex, is required during development to specify the identity of parasegments 10-14. Based on genetic studies, Casanova, J., Sánchez-Herrero, E. and Morata, G. (1986) Cell 47, 627-636, proposed that the Abd-B gene consists of two distinct elements that provide a morphogenetic (m) function in PS 10-13 and a regulatory (r) function in PS 14, where it represses m function. Here we present molecular confirmation of this genetic model. Using specific antibodies, we show that the 55 X 10(3) M(r) and 30 X 10(3) M(r) Abd-B proteins, predicted by cDNA analysis, are indeed present in PS 10-13 and PS 14, respectively. We also examine Abd-B mRNA and protein expression patterns in embryos mutant for either the m or r function. These data allow us to unambiguously assign m function to the 55 X 10(3) M(r) protein and r function to the 30 X 10(3) M(r) protein. Furthermore, as postulated by the model, transcription of the mRNA encoding the m protein is derepressed in PS 14 in the absence of r function. We have also studied the effect of mutations mapping in the infra-abdominal (iab) region located downstream of the Abd-B gene. Genetic studies suggest that the iab region contains cis-acting regulatory elements controlling Abd-B expression in PS 10-12. We present molecular evidence for the presence of downstream cis-regulatory elements by analyzing Abd-B mRNA and protein patterns in iab-6 and iab-7 embryos. Our analysis reveals the presence of parasegment and cell-specific regulatory elements of the Abd-B gene within each iab region. The Abd-B gene may provide a model for the understanding of similarly complex homeotic genes in higher organisms.

Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex.

The infra-abdominal (iab) regions of the biothorax complex are thought to cis-regulate expression of the abdominal-A (abd-A) and Abdominal-B (Abd-B) transcripts. These large cis-regulatory regions are also actively transcribed. Here we present a detailed analysis of the transcription products of the iab-4 region. During early embryogenesis a 6.8-kilobase (kb) RNA is transcribed and processed to yield 1.7- and 2.0-kb poly(A)+ RNAs. These RNAs are expressed from the time of cellular blastoderm formation to germ-band shortening and are localized in parasegments 8-14 (the posterior second through the anterior ninth abdominal segments). Sequence analysis of the two RNAs suggests that they may not be translated. We discuss some possible functions of the 1.7/2.0-kb iab RNAs and the significance of their similarity to the bithoraxoid (bxd) RNAs and other transcripts from the iab regions of the abd-A and Abd-B domains.

The morphogenetic and regulatory functions of the Drosophila Abdominal-B gene are encoded in overlapping RNAs transcribed from separate promoters.

The Abdominal-B (Abd-B) gene of the Drosophila bithorax complex is a homeotic gene with two subfunctions: the morphogenetic element required for specifying the identity of parasegments (PS) 10-13 and the regulatory element that represses the expression of other homeotic genes in PS14. Here, we provide evidence that four classes of overlapping transcripts are generated from the Abd-B gene and characterize three of the transcripts in detail. We determined the transcription initiation sites for the 4.6- and 3.4-kb RNAs and show that they are generated from separate promoters. Both of these transcripts are present throughout the period during which the Abd-B subfunctions are required. A mutation that inactivates the morphogenetic function is associated with a 411-bp deletion of the initiation site for the 4.6-kb RNA. The regulatory function mutations disrupt the transcription unit for the 3.4-, but not the 4.6-kb, RNA. These results support the assignment of the morphogenetic function to the 4.6-kb RNA and the regulatory function to the 3.4-kb RNA. A 7.8-kb RNA expressed during embryogenesis may also contribute to the regulatory function. Sequence analysis of cDNAs indicates that the 4.6-kb RNA encodes a 55-kD protein (the m protein), whereas the 3.4-kb RNA encodes a 30-kD protein (the r protein). The m and r proteins share a carboxy-terminal sequence that includes the homeo domain, but the r protein lacks a glutamine-rich amino-terminal domain found in the m protein.

Contact points between a positive transcription factor and the Xenopus 5S RNA gene.

We have investigated the contact points of a positive transcription factor with the internal control region of the 5S ribosomal RNA genes of Xenopus. The methylation of any one of eight G residues clustered at the 3' end of the internal control region on the noncoding strand of the DNA or the ethylation of their surrounding phosphates interferes with the binding of this protein. The importance of the 5' half of the control region is shown by a binding affinity of the protein to oocyte 5S RNA genes that is one fourth that to somatic 5S RNA genes. This can be attributed to two base changes at residues 53 and 55 in the 5' part of the control region. The different affinities of the transcription factor for the oocyte and somatic 5S RNA genes may contribute to their differential expression in somatic cells. The almost exclusive location of strong contact points on the noncoding strand of the gene suggests how transcription events could proceed repeatedly along a gene that is assembled into a stable transcription complex. It is proposed that the complex is transiently shifted to the noncoding strand as each round of RNA synthesis occurs from the coding strand.

The binding of a transcription factor to deletion mutants of a 5S ribosomal RNA gene.

A transcription factor binds specifically to a region within the 5S ribosomal RNA gene that is known to be required for accurate initiation of transcription of the gene. We have confirmed the significance of this interaction by comparing various deletion mutants for their ability to initiate transcription and to bind the protein. Loss of specific binding of the protein occurs when the gene is deleted beyond +83 from the 3' direction. This coincides exactly with the loss of ability to initiate transcription. In contrast, deletions entering the gene from the 5' direction and extending beyond the 5' border of the control region (+50/+55) give partial protection of the remainder of the control region. This observation can explain why deletions beyond the 5' border of the control region that are unable to initiate transcription can still compete for the transcription of the wild-type 5S RNA gene.

A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region.

The 5S ribosomal RNA gene of Xenopus contains a region within the gene that directs the initiation of 5S RNA synthesis. This result was obtained by enzymatically deleting a fragment of X. borealis somatic 5S DNA from the 5' side of the gene and cloning the resultant mutants. These deletion mutants were tested for their ability to support 5S RNA transcription in an oocyte nuclear extract. Mutants lacking the entire 5' flanking region synthesized 5S RNA or slightly smaller RNAs that were initiated a few nucleotides into the gene. Mutants deleted as far as 50 nucleotides into the gene synthesized discrete RNAs of 116--121 nucleotides. These RNAs were fused transcripts that were initiated in the plasmid vector, transcribed through the remainder of the 5S RNA gene and terminated at the end of the gene. Mutants deleted 55 or more nucleotides into the gene synthesized little or no 5S size RNA. When additional nucleotides were inserted between nucleotides +40 and +41 of the gene, discrete transcripts of approximately 120 nucleotides were synthesized that had initiated within the gene. We conclude that a control region within the gene directs RNA polymerase III to initiate transcription approximately 50 nucleotides upstream from the 5' border of this region. The 3' border of the control region resides between gene residues +80 and +83 as determined in work described in the accompanying paper (Bogenhagen, Sakonju and Brown, 1980). When this control region is present, the exact site of initiation is determined by the sequence around the start site.