Department of Biology, Gilmer Hall, University of Virginia, P.O. Box 400328, Charlottesville, VA 22904-4328, USA
*Author for correspondence (e-mail: crc2s{at}virginia.edu)
Accepted September 5, 2001
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SUMMARY |
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Key words: bHLH, Autoregulation, Oogenesis, Insulation, daughterless, Drosophila
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INTRODUCTION |
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The helix-loop-helix (HLH) family of transcription factors includes over 240 different proteins that are present throughout eukaryotes, including both ubiquitous and temporally/spatially restricted transcription factors (Massari and Murre, 2000). HLH proteins dimerize via amphipathic helices and interact directly with the major groove of DNA via a basic domain. These proteins fall into seven specific classes based on dimerization capabilities, tissue distribution and DNA target specificity (Murre et al., 1994). Class I HLHs, also known as E proteins, can form either heterodimers or homodimers, are widely expressed, and have DNA binding specificity for the E box (Ephrussi et al., 1985). The more numerous class II HLHs heterodimerize with class I HLHs and show tissue-restricted expression patterns and target sequence specificity that varies with different heterodimer partners and their conformation (Kophengnavong et al., 2000). In vertebrates, class I HLHs are essential for commitment to the B lymphoid lineage (Bain et al., 1994; Zhuang et al., 1994), T cell development (Barndt et al., 2000), regulation of V(D)J recombination (Romanow et al., 2000), muscle differentiation (Lassar et al., 1991) and expression of differentiated cell products such as insulin (Sharma et al., 1997). This partial list of vertebrate class I HLH functions does not include cases of transcriptional regulation for which a class II HLH is known, but its requisite class I partner has yet to be identified.
In Drosophila, there is only one class I HLH protein, encoded by the daughterless (da) gene, and this distinction not only reflects its apparent ubiquitous expression, but also accounts for the large number of developmental processes in which it functions (Cronmiller and Cummings, 1993; Massari and Murre, 2000; Moore et al., 2000). In the embryo, Da protein is required for the early transcriptional activation of Sex lethal (Sxl) during sex determination (Cronmiller and Salz, 1994; Keyes et al., 1992), for differentiation of the mesoderm (Gonzalez-Crespo and Levine, 1993), and for the establishment of the proneural field that gives rise to the central and the peripheral nervous systems (Caudy et al., 1988a). In larvae, da is again required for establishment of the neural field for adult sensory organ precursers (Modolell, 1997), for progression of the morphogenetic furrow of the developing eye (Brown et al., 1996) and for the differentiation of the salivary gland (King-Jones et al., 1999). In adults, da is required for ovarian follicle formation (Cummings and Cronmiller, 1994). Countless additional developmental functions have been implied, based on tissue culture experiments, identification of genes encoding tissue-restricted class II HLHs, and conservation of developmental processes requiring E proteins in other organisms.
It is generally believed that Da accomplishes its numerous discrete developmental roles through collaboration with regulated HLH binding partners. During sex determination, Da associates with Sis-b/Sc (sisterless-b/scute), which is present in the early embryo at high enough levels to activate the Sxl early promoter only in females (Deshpande et al., 1995; Yang et al., 2001). Da associates with HLH proteins from the neural-specific Achaete-Scute Complex in establishment of the proneural field (Cabrera and Alonso, 1991). For the formation of multiple dendritic neurons in the peripheral nervous system, Da heterodimerizes with Amos, which is present in patches of ectodermal cells and soon thereafter is restricted to sensory organ precursors (Huang et al., 2000).
The Da protein, however, is not simply a generic unregulated binding partner for other developmentally regulated HLH proteins. Through analysis of a unique female sterile allele, we have discovered precise transcriptional regulation of da. And, at least in the ovary, either reducing or increasing the amount of Da causes distinct mutant phenotypes. Thus, among cells that contain both Da and its relevant Class II binding partner, variable Da levels may restrict the formation of functional transcriptional complexes, indicating regulatory specificity dictated by da.
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Materials and Methods |
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Suppressors of the dalyh female sterility were isolated after standard ethyl methanesulfonate mutagenesis of males (Lewis and Bacher, 1968). Three independent dominant suppressors, including Su(lyh)26H6, were recovered and subsequently mapped to the fourth chromosome.
Transvection experiment
Ovaries from flies of the genotype C(1)A/Tp(2;Y)cb50; dalyh stla16/Df(2L)J27 were examined. Standard polytene salivary gland squashes were carried out to examine chromosomes of male larvae carrying Dp(2;Y)B231; no pairing was seen between the Y duplication of the da region and the second chromosome.
Staining
Ovaries were fixed and DAPI stained as previously described (Cummings and Cronmiller, 1994). Whole-mount ovary immunohistochemistry was performed as previously described (Cronmiller and Cummings, 1993) with monoclonal mouse anti-Hts (1B1) (Zaccai and Lipshitz, 1996) (1:10 dilution) and polyclonal rabbit anti-Vasa (Styhler et al., 1998) (1:1000 dilution) using FITC- or TRITC-conjugated secondary antibodies (Jackson Immunoresearch).
Molecular analyses
Standard molecular techniques were used except where otherwise noted (Sambrook et al., 1989).
PCR
To amplify and identify the transposon insertion in dalyh, PCR was performed using DyNAzyme EXT (Finnzymes) with primers 0A (5'-GGCTCAACGTCAACACTCGCTGCAAC-3') and P1B (5'-CGTACATAAGGCTGTATACGCACGG-3'). The PCR product was cloned into the pGEM T-easy vector (Promega).
DNA sequence analysis
Sequence from both ends of the dalyh springer insertion was obtained (Accession Numbers AF418012 and AF418013). A full-length springer contig of 7509 bp was constructed from unordered sequenced fragments from the Berkeley Drosophila Genome Project (BAC clones D849, D848, D841 and D823) using NCBI Blast, MacVector and AssemblyLIGN (Oxford Molecular Group). The restriction pattern roughly resembles that of the originally defined 8.8 kb springer element (Karlik and Fyrberg, 1985). Three full-length springer elements are included in the completed genome project sequence (GenBank Accession Numbers AE003580, AE003776 and AE003433) (Adams et al., 2000). Transcription factor binding sites within the da region were analyzed with MatInspector and the TransFac database (Quandt et al., 1995; Wingender et al., 2000).
Northern blots
PolyA+ mRNA was loaded (5 µg per lane). Hybridization was carried out using UltraHyb (Ambion) at 42°C. 32P-labeled probes were prepared by random priming of da cDNA MN6, da genomic fragment 5 and neighboring fragment 6 (to detect Mdh1) (Cronmiller et al., 1988), and EST LP12271 (to detect rp49). Band intensities were quantified using PhosphorImager Scanner and ImageQuant software (Molecular Dynamics).
Real-time RT-PCR
Four- to 6-day old female flies, carrying two copies of the phsp70-da+ transgene and one copy of the pda-gal4=da.G32 transgene, were treated with heat shock in a 37°C water bath or kept at room temperature for 3 hours. RNA was extracted immediately after treatment from 20 flies under each condition in duplicate using TRIzol reagent (Life Technologies) as per the manufacturers protocol, followed by treatment with RQ1-DNase (Promega) and repurification with TRIzol reagent. Amplification reactions were prepared in triplicate using the Access RT-PCR System (Promega), with SYBR Green detection in a Cepheid SmartCycler (Morrison et al., 1998). Conditions used were 48°C for 45 minutes; 94°C for 2 minutes; 40 cycles of 94°C for 10 seconds, 53°C for 15 seconds and 68°C for 20 seconds. Primer pairs: galRT1a (5'-TAACCGTCCACCCTCTCGTAACTC-3') and galRT1b (5'-AAAAGGCGTGACTGAGCGATGC-3'); or mdh1a (5'-TACCATTGGCGGTCACCTTG-3') and mdh1b (5'-TCATTATTTGGGGCAACCACTC-3'). Melting curves were analyzed for purity of product.
Statistical analysis
Conservatively, as RT-PCR reactions within RNA preps were not independent, the means of each RNA prep for each transcript were compared; no significant difference was seen between RNA preps for Mdh1 under either treatment or for gal4 with heatshock, although a significant change was seen between RNA preps for gal4 without heat shock (P<0.05). To increase the power of statistical analysis each observation was then treated as an independent data point. A t test was used on gal4 or Mdh1 transcripts to test for differences between heat shock treatment (yes, no) using SAS v8.0 (SAS Institute, Cary, NC).
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RESULTS |
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Da protein expression in dalyh mutants is also disrupted specifically in the ovary. Staining of wild-type ovaries showed clear nuclear Da protein, while dalyh mutant ovaries showed no nuclear localized protein within the ovariole (Fig. 2A). Western blots of ovary extracts detected Da protein but at reduced levels (data not shown). The protein seen in ovary extract probably corresponded to other Da-containing tissues included in the extract: the epithelial sheath that surrounds each ovariole and its associated muscles, the tracheae that infiltrate the ovary and the oviduct.
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dalyh alters levels of da transcription
The mechanism of da gene disruption by the springer element is different than other analyzed springer alleles. Molecularly characterized springer-induced mutations (Tm23, Nfa3, f36a, Mhc2, Mhc3) each result from insertion within exons or near alternatively used exons to produce aberrant transcripts usually with premature transcriptional termination within the springer LTR (Davis et al., 1998; Hoover et al., 1993; Ishimaru and Saigo, 1993; Karlik and Fyrberg, 1985; Kidd and Young, 1986). We tested whether dalyh produced any novel transcripts by probing Northern blots with a full-length da cDNA probe or a genomic fragment that spanned the intron and included part of both exons (Fig. 2B). Both probes exclusively detected the two known da transcripts (Caudy et al., 1988b; Cronmiller et al., 1988), but the dalyh mutation increased the da transcript levels. Normalized to the mRNA of the nearby Mdh1 gene, which encodes malate dehydrogenase, da transcript levels were 1.5- and 1.9-fold higher (males and females, respectively) in dalyh than in wild-type flies (Fig. 2D). Comparable increases were estimated when transcripts were compared with an rp49 control (data not shown).
Taken together, the RNA and protein analyses suggest that the dalyh springer insertion acts as a transcriptional insulator. The absence of Da protein in dalyh mutant ovaries must result from blocked da expression in somatic cells: in this mutant, da is expressed everywhere except in the somatic ovary. Such a loss of da mRNA would not be apparent in whole fly mRNA; even in ovaries specifically, loss of somatic mRNA would be concealed by strong germline expression of da, whether assayed by northern blot or in situ hybridization (Cummings and Cronmiller, 1994). Consistent with this interpretation, we sequenced the entire da-coding region of dalyh and found no additional changes (data not shown); thus, post-transcriptional loss of da product was ruled out. Additionally, the springer element also appears to insulate a negative regulatory element that results in an overall increase in da transcription, hence the elevated mRNA levels evident on the northern blots.
Genetic evidence for da autoregulation
Surprisingly, in genetic interaction tests dalyh does not behave like a genetic null: ordinarily, the da somatic ovary function is particularly sensitive to gene dose, such that a da loss-of-function allele exhibits second site non-complementation with mutations in other genes involved in follicle morphogenesis (Cummings and Cronmiller, 1994; Grammont et al., 1997). By contrast, dalyh did not. A specific example of this paradoxical genetic behavior was the interaction we observed between da and stall (stl), another gene required for follicle formation (Schupbach and Wieschaus, 1991). A null allele of da completely failed to complement the null allele, stla16; ovaries of doubly heterozygous females had no normal ovarioles (Fig. 3A). Even the hypomorphic das22 allele failed to complement stla16, with 65% of the ovarioles having defects (Fig. 3B). However, dalyh fully complemented stla16; no defects were seen (Fig. 3C). Furthermore, we tested 46 chromosomal deletions with which the null allele, da2, had exhibited dominant interactions to produce mutant ovary phenotypes (J. E. S. and C. C., unpublished); all produced completely normal ovaries in combination with dalyh (data not shown). Thus, the da+ chromosome of the heterozygous dalyh genotype appeared to induce wild-type function from its mutant dalyh homolog, which we showed did not produce Da protein in the somatic ovary.
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Further genetic evidence in support of transcriptional autoregulation is the discovery that ectopic da+ can convert the dalyh homozygous loss-of-function phenotype to a gain-of-function phenotype. We could observe only a subtle gain-of-function phenotype associated with an increased dose of da+ when we generated flies that had three copies of the gene, the extra copy being provided by a chromosomal duplication (either in tandem or by transposition). Although the ovaries from these flies had properly formed follicles, we occasionally observed interfollicular stalks that were distinctly longer than normal (Fig. 4B). In terms of the nature of this phenotype, it was not surprising that excess da+ could lead to these longer stalks, because reduced da+ resulted in loss of stalks. When we added an ectopic copy of da+ to the homozygous dalyh genotype, we saw dramatic gain-of-function phenotypes that were similar to, but also more extreme than, those observed in the 3X-da+ ovaries. For example, in addition to long interfollicular stalks, we found ovarioles with shrunken germaria that occasionally were attached directly to mid-to-late stage follicles (Fig. 4C-E). Although the expressivity of these phenotypes varied, they were unlike any phenotypes that resulted from da loss of function. The conversion of dalyh from a loss-of-function to a gain-of-function phenotype was not dependent upon the specific translocation, as two copies of a heat-inducible da+ transgene completely rescued the loss-of-function phenotype at 25°C and produced the gain-of-function phenotype when adults were placed at 32°C (Fig. 4F). These da overexpression phenotypes were also not dependent upon the dalyh allele, as we could phenocopy such overexpression defects with the heat-inducible da+ transgenes in an otherwise wild-type background using 30 minute 37°C pulses every 6 hours (Fig. 4G). Thus, the dalyh-associated overexpression phenotype resulted when wild-type Da protein transactivated the mutant dalyh alleles, enabling them to produce their own wild-type da product. Furthermore, once transcriptionally activated, each dalyh allele produced a greater amount of that product, consistent not only with the more extreme overexpression phenotype of the allele, but also with its overall increased mRNA levels.
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DISCUSSION |
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In wild-type flies, several distinct transcriptional controls provide for a tightly regulated level of da expression within the somatic ovary (Fig. 7A). Initiation of da transcription requires an enhancer within the single intron of the gene. After activation of da transcription, the Da protein itself functions to maintain da expression. Indeed, since multiple canonical E-boxes (Ephrussi et al., 1985) are present in the da promoter region, this autoregulatory function may result from a direct interaction of Da protein with its own regulatory sequences. Finally, negative cis-acting sequences downregulate da transcription, thus preventing autoregulatory da expression from escalating to produce deleteriously high levels of Da protein. Such deleterious levels are achieved by overexpression of heat shock inducible da+ transgenes.
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Based on the failure of transcription of dalyh in the somatic ovary and from an analysis of a da promoter fusion transgene, we have identified a STAT (Drosophila Stat92E)-binding site as a candidate for the cis-acting enhancer. STAT (signal transducers and activators of transcription) proteins are activated by tyrosine kinases in response to cytokine or growth factor signals and play essential developmental roles in growth and differentiation, and they are constitutively activated in many cancers (Bromberg, 2001; Horvath, 2000). The Stat92E binding site in da (TTCATGGAA) is the only predicted transcription factor-binding site found exclusively downstream of the springer insertion and within the extents of the da.G32 reporter. The somatic ovary enhancer must be included in the da.G32 reporter (Fig. 2B), as this transgene is expressed in the ovary, even in dalyh mutants (data not shown). Moreover, a temperature sensitive loss-of-function allele of Stat92E shows a da-like mutant ovary phenotype (K. Baksa and C. Dearolf, personal communication). We propose that Stat92E is essential for the initiation of da transcription within the somatic ovary.
Da protein appears to be necessary for maintenance of its own transcription, and the simplest molecular model for da autoregulation is direct transcriptional activation. Although Da homodimers can bind DNA in vitro (Murre et al., 1989) and the mammalian homolog, E47, does function as a homodimer in B cell development (Shen and Kadesch, 1995), there are no examples of Da protein homodimerizing to activate transcription in vivo. More likely, Da acts on its own promoter by collaboration with another bHLH-binding partner. We have identified several possible candidate binding partners, based on ovary phenotypes in genetic interaction tests (E. Basler and C. C., unpublished). For example, one candidate is achaete (ac), which transcriptionally autoregulates during the development of sensory bristles in wing imaginal discs (Van Doren et al., 1992). For this process, Ac protein heterodimerizes with Da; perhaps they collaborate again in the somatic ovary with the da gene as their target.
We have identified two cis-acting elements involved in da transcriptional regulation in the ovary, but there are likely to be more cis-regulatory elements whose use may differ between or within other tissues. Although nearly ubiquitous throughout development, Da protein is present at significantly different levels in various tissues, or even within individual tissues. For example, the CNS includes cells with levels of Da that range from very low to very high (Cronmiller and Cummings, 1993), and in eye discs, dynamic changes in Da protein levels correlate with the progression of the morphogenetic furrow (Brown et al., 1996). If Da protein levels directly reflect da transcript levels, these observations suggest that precise regulation of da is crucial for developmental processes, and the regulatory sites identified are probably not sufficient to account for the scope of regulation necessary. The da.G32 reporter, which shows a mottled expression pattern that is not attributable to position effect variegation (data not shown), indicates that this transgene is missing crucial binding sites for regulatory factors. Additionally, this construct is unable to rescue embryonic lethality when driving a Gal4-dependent da+ transgene (Giebel et al., 1997). However, a 15 kb genomic da+ transgene that includes the da.G32 regulatory region and an additional 12 kb downstream rescues da mutant flies to adulthood (H. Vaessin, personal communication); this construct may contain all of the necessary da regulatory sequences. Thus, da expression, like that of many other protein-coding genes, is dependent upon a balance of multiple positive and negative regulators (Lee and Young, 2000).
dalyh identifies springer as an insulator element
The dalyh allele is the first springer-induced mutation in which this retrotransposon is documented to disrupt gene function by acting as an insulator; this discovery emphasizes the similarity between springer and the extensively-characterized gypsy retrotransposon. Like gypsy (Corces and Geyer, 1991), springer can disrupt gene function in two ways: either by altering the normal transcripts of a gene or by acting as an insulator. All other springer-induced alleles whose expression has been characterized to date produce aberrant transcripts (Davis et al., 1998; Hoover et al., 1993; Ishimaru and Saigo, 1993; Karlik and Fyrberg, 1985; Kidd and Young, 1986). This newly discovered similarity between springer and gypsy prompted us to look for Su(Hw)-binding sites in springer, as Su(Hw) mediates gypsy insulation (Roseman et al., 1993). Springer has no Su(Hw)-binding sites, so it must be bound by a different insulating protein.
Su(lyh) may encode the springer insulator protein. Su(lyh) dominantly suppresses the dalyh insulation of both the cis-acting enhancer and the cis-acting negative regulator. The dominant nature of this suppression may be unique to the da locus, as autoregulation will amplify even the small amount of da gene product that results when the insulation is only weakly suppressed. However, su(Hw) can act as a dominant suppressor of some gypsy-induced alleles. (Hoover et al., 1992). Suppression of the dalyh mutant phenotype is completely penetrant when the insulator protein is eliminated altogether, as in the case of Su(lyh) homozygotes. We expect that the Su(lyh) protein will function like Su(Hw) protein by binding specific sites within springer.
In addition to Su(lyh), other previously described insulator proteins may be involved in springer insulation. For one, the zeste-white 5 (zw5) gene product binds to specific sequences within the scs chromatin domain boundary of the 87A7 heat shock locus (Gaszner et al., 1999), and there are two of these binding sites in springer. For another, BEAF binds to a clustered CGATA array within several chromatin boundaries (Cuvier et al., 1998), including the boundary of scs, and there are 12 unclustered CGATA sequences in springer. Finally, proteins of the polycomb and trithorax groups appear to collaborate with Su(Hw) in gypsy insulation (Gerasimova and Corces, 1998), and there is a polycomb response element (Mihaly et al., 1998) in springer. Future work will clarify what role, if any, these factors play in springer-associated insulation.
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ACKNOWLEDGMENTS |
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