Abstract
mod(mdg4), also known as E(var)3-93D, is involved in a variety of processes, such as gene silencing in position effect variegation (PEV), the control of gypsy insulator sequences, regulation of homeotic gene expression, and programmed cell death. We have isolated a large number of mod(mdg4) cDNAs, representing 21 different isoforms generated by alternative splicing. The deduced proteins are characterized by a common N terminus of 402 amino acids, including the BTB/POZ-domain. Most of the variable C termini contain a new consensus sequence, including four positioned hydrophobic amino acids and a Cys2His2 motif. Using specific antibodies for two protein isoforms, we demonstrate different distributions of the corresponding proteins on polytene chromosomes. Mutations in the genomic region encoding exons 1–4 show enhancement of PEV and homeotic transformation and affect viability and fertility. Homeotic and PEV phenotypes are enhanced by mutations in other trx-group genes. A transgene containing the common 5′ region of mod(mdg4) that is present in all splice variants known so far partially rescues the recessive lethality of mod(mdg4) mutant alleles. Our data provide evidence that the molecular and genetic complexity of mod(mdg4) is caused by a large set of individual protein isoforms with specific functions in regulating the chromatin structure of different sets of genes throughout development.
DURING development of multicellular organisms, the spatial and temporal expression of many genes must be precisely regulated. Growing evidence implicates changes in chromatin structure as an important level in this control. Although the organization of the nucleosome is well understood (Lugeret al. 1997), we lack substantial knowledge of higher-order chromatin structure. Chromatin proteins involved in structural changes during transcriptional regulation were identified by several approaches. In addition to biochemical analysis of chromatin remodeling complexes (Tsukiyama and Wu 1997; Kadonaga 1998), genetic dissection of chromatin structure has been performed successfully in Drosophila.
Several genes involved in the regulation of chromatin structure were identified as regulators of homeotic gene complexes. The Polycomb group of genes has been shown to be essential for silencing of homeotic genes outside their limits of expression. This function involves the formation of large multisubunit protein complexes, which limit the access of transcription factors to the genes to be silenced (Paro and Harte 1996; Pirrotta 1997). Trithorax (trx) group genes, on the other hand, encode positive regulators of homeotic gene expression that in some way counteract the effect of the Polycomb group silencers (Simon 1995).
Modification of position effect variegation (PEV) provides another powerful approach to identifying genes involved in the regulation of chromatin structure (for review see Reuter and Spierer 1992; Weiler and Wakimoto 1995; Wallrath 1998). More than 100 genes that, when mutated, either suppress or enhance PEV have been identified. The hypothesis that mutations in suppressors of PEV affect components of chromatin complexes involved in silencing is consistent with the molecular and cytological data available for this class of proteins (Wallrath 1998). The enhancers of PEV, which exert opposite effects, are less well studied. Insertional PEV enhancers have been isolated after remobilization of modified P-element transposons (Dornet al. 1993a). The first enhancer gene of PEV was molecularly characterized using the E(var)3-93Dneo129 mutation (Dornet al. 1993b). In a study of modifiers of gypsy-induced mutations, it was shown that mutations in mod(mdg4) are also enhancers of PEV and are allelic to the E(var)3-93Dneo129 mutation (Gerasimovaet al. 1995; Gerasimova and Corces 1998). Trithorax-like (Trl), the second molecularly characterized PEV enhancer gene, encodes the GAGA factor (Farkaset al. 1994). Both genes encode transcriptional regulators and mutations display phenotypes characteristic of the trx group genes. Molecular analysis of both mod(mdg4) and Trl demonstrated the existence of several transcripts generated by alternative splicing (Dornet al. 1993b; Gerasimovaet al. 1995; Benyajatiet al. 1997; Harveyet al. 1997). By immunocytology, proteins of both genes were shown to be associated with many sites along the polytene chromosomes. Analysis of two individual Trl isoforms revealed a complete colocalization and their physical interaction was demonstrated by coimmunoprecipitation (Benyajatiet al. 1997).
Six different Mod(mdg4) isoforms have been identified so far (Dornet al. 1993b; Gerasimovaet al. 1995; Harveyet al. 1997). All of them contain a common N terminus, including the BTB/POZ domain, which is a conserved protein-protein interaction domain present in a number of genes involved in regulation of transcription (Bardwell and Treisman 1994; Ahmadet al. 1998). In most cases, the C terminus of such BTB/POZ-domain proteins contains one or more zinc finger motifs of the Cys2His2 type. Their involvement in DNA binding could be shown for two BTB zinc finger proteins, Trl and Tramtrack (Biggin and Tjan 1988; Harrison and Travers 1990).
Recently, we demonstrated genetic interactions between mod(mdg4) and several suppressors of PEV, suggesting that they were involved in a common process in the regulation of chromatin structure. Similarly, genetic interaction between mod(mdg4) mutations and mutations in trx group genes could be shown (Dornet al. 1993b; Gerasimova and Corces 1998), indicating that the set of genes identified as homeotic or PEV regulators may functionally overlap. Because of the ability of mod(mdg4) alleles to modify the insulating effect of gypsy transposon insertions, Gerasimova et al. (1995) proposed a general role for mod(mdg4) in the control of chromatin insulators. Doom, another splice variant of mod(mdg4), was identified as an interactor of the baculovirus inhibitor of apoptosis protein (IAP), whose involvement in apoptosis could be demonstrated directly (Harveyet al. 1997). Together, these data demonstrate a considerable molecular and functional complexity for the mod(mdg4) gene.
In this article, we describe a comprehensive molecular analysis of mod(mdg4). We have identified 17 additional splice variants, that reflect the complexity of Mod-(mdg4) protein expression seen on Western blots. The alignment of the deduced protein isoforms revealed the existence of a new conserved protein consensus motif in addition to the BTB/POZ domain in most of these isoforms. By the use of isoform-specific antibodies, we provide the first evidence for differential binding of Mod(mdg4) protein isoforms on polytene chromosomes. Genetic and molecular analyses of mutations demonstrate an essential maternal contribution of mod-(mdg4) to early embryonic development. Significantly reduced amounts of Mod(mdg4) proteins in egg chambers of mutant females result in maternal effect lethality.
MATERIALS AND METHODS
Fly stocks and genetic analysis: Fly stocks were maintained under standard conditions. Chromosomes and mutations are described in Lindsley and Zimm (1992). All crosses, unless otherwise indicated, were carried out at standard temperature (25°). Isolation of mod(mdg4) mutant alleles is described in Dorn et al. (1993b).
The degree of homeotic transformation of abdominal segment A5 to A4 in males was quantified using arbitrary units. Flies were graded into seven classes on the basis of the size of the area without black pigmentation on A5. Unpigmented A5 equals grade 6; grade 5 corresponds to an unpigmented area of ~80%; grade 4 equals ~50% of unpigmented A5; ~30% unpigmented A5 was given grade 3; grade 2 represents A5 with isolated medium-sized unpigmented patches; A5 with small unpigmented spots corresponds to grade 1; and grade 0 represents completely pigmented A5. Quantification of PEV in wm4 was performed by pigment measurement as described in Ephrussi and Herold (1944). The mutant alleles AI117 and AI351 obtained from Dr. M. Frasch were renamed mod-(mdg4)06 and mod(mdg4)07, respectively.
Standard procedures: Restriction analysis, subcloning, and Southern blot analysis were performed according to Sambrook et al. (1989). Genomic DNA from adult flies was isolated as described in Jowett (1986). Hybridization probes were generated by random priming using the Multi Prime labeling kit (Amersham, Piscataway, NJ).Forradioactive labeling,[β-32P]dATP was used. Sequencing was performed using the T7 sequencing kit (Pharmacia, Piscataway, NJ) and [β-35S]dATP for radioactive labeling or alternatively by cycle sequencing and analysis using the sequencer ABI 377 (Perkin Elmer, Norwalk, CT).
RNA isolation and analysis: RNA was isolated as described by Auffray and Rougeon (1980). The mRNA purification kit (Pharmacia) was used to purify poly(A)+ RNA. RNA was hybridized in a solution containing 50% formamide at 42° after size fractionation on 1% agarose-formaldehyde gels and transfer to Hybond-N+ (Amersham).
P-element-mediated germline transformation: Germline transformation was performed as described in Rubin and Spradling (1982). The 7.5-kb BamHI genomic fragment containing the 5′ region of mod(mdg4) was inserted in the transformation vector pW8 (Klemenzet al. 1986). One second chromosomal transgenic line was obtained. Mobilization of the insertion using TM3, Sb Δ2-3 as a transposase source resulted in two additional lines with the w+ insert on a second chromosome carrying the dominant marker Sco. All three transgenic lines were able to rescue the recessive lethality of mod(mdg4)02. For complementation crosses in Table 3 and Figure 9, line P(w+7.5kb BamHI)-2/1 was used.
Screening of embryonic cDNA libraries: cDNA clones have been isolated from two embryonic libraries: 12- to 24-hr embryonic cDNA library in λgt10 (Pooleet al. 1985) and 2- to 12-hr embryonic cDNA library in λZAPII (Stratagene, La Jolla, CA). The 0.5-kb genomic SalI fragment that is colinear to the earlier identified cDNA clones mod(mdg4)-58.0 (23gt) and mod(mdg4)-67.2 (38gt) (Dornet al. 1993b) was used as a radioactive labeled probe. Isolated clones were analyzed by restriction analysis and partial sequencing. Some of the isolated clones contained sequences at the 5′ ends derived from other cloned genes, indicating that artificial clones resulted during construction of the libraries. The numbers of independent cDNA clones isolated were as follows:
one cDNA clone: mod(mdg4)46.3, 51.4, 52.0, 56.9, 57.4, 60.1, 65.0
two to four cDNA clones: mod(mdg4)53.1, 54.2, 54.6, 55.1, 55.3, 55.7, 56.3, 58.0, 58.6, 59.0, 62.3, 64.2
five to eight cDNA clones: mod(mdg4)55.6, 67.2.
The GenBank/EMBL numbers of the representative mod-(mdg4) cDNA clones are AJ277174–AJ277194.
Library construction and screening: Genomic libraries were constructed from partially Sau3A-digested and size-fractionated genomic DNA prepared from heterozygous wm4; mod(mdg4)02/TM3, Sb Ser and wm4; mod(mdg4)03/TM3, Sb Ser mutant flies using the λZAPII cloning system (Stratagene). Screening and phage DNA purification was performed according to the manufacturer's protocol. The 7.2-kb genomic BamHI fragment was used as a probe for screening.
Antibodies: The whole open reading frame (ORF) of the cDNA clone mod(mdg4)-58.0 as well as the specific C-terminal parts of the clones mod(mdg4)-58.0 [amino acids (aa) 403–534] and mod(mdg4)-67.2 (aa 403–610) were introduced into the unique BamHI site of the expression vector pGEX-2T (Pharmacia). The proteins were expressed as fusion protein with glutathione-S-transferase of Schistosoma japonicum (Smith and Johnson 1988) and purified from inclusion bodies via urea extraction (Küpperet al. 1982) or as soluble proteins via affinity chromatography using glutathione-Sepharose as matrix. Antibodies were generated in rabbits and mice (Eurogentec). The polyclonal rabbit antibody anti-Mod(mdg4)-58.0403-534 was purified on an affinity column with the corresponding recombinant protein.
Western blot analysis and immunoprecipitation: Nuclear extracts from 0- to 12-hr Drosophila embryos were prepared according to Elgin and Hood (1973). Nuclei were lysed in SDS protein gel loading buffer and incubated at 95° for 5 min.
For immunoprecipitation, 4 g of dechorionated embryos were homogenized in ice-cold 50 mm HEPES, pH 7.6; 385 mm NaCl; 0.1% Tween 20; 0.1% EGTA; 1 mm MgCl2; 0.1 mg leupeptin, pepstatin, and aprotinin; 1 mm phenylmethylsulfonyl fluoride (PMSF) and incubated for 15 min at 4°. All subsequent steps were performed at 4°. After centrifugation for 1 hr at 15,000 × g, 10% glycerol was added to the supernatants, and the extracts were frozen in liquid nitrogen and stored at −70°. Embryonic extracts containing 15 mg protein were precleared with 25 μl 50% protein-A-Sepharose in binding buffer (50 mm HEPES, pH 7.6; 192.5 mm NaCl; 0.1% Tween 20; 7 mm DTT; 10% glycerol; 1 mm PMSF) for 1 hr. After centrifugation at 15,000 × g for 30 min, 1 μg affinity-purified anti-Mod(mdg4)-58.0403-534 antibody was added to the supernatant, and the extracts were incubated for 2 hr. A total of 50 μl of 50% protein-A-Sepharose was added for binding of the complexes for 2 hr. The beads were washed four times for 10 min in 1 ml washing buffer (50 mm HEPES, pH 7.6; 150 mm NaCl; 0.1% Tween 20; 7 mm DTT; 10% glycerol; 1 mm PMSF) and resuspended in 30 μl SDS protein gel loading buffer. After incubation at 95° for 5 min, the eluted complexes were separated on a SDS-PAGE gel (Laemmli 1970).
After electrophoresis, the proteins were electroblotted to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% nonfat dry milk in PBS for at least 1 hr. The primary antibodies [anti-Mod(mdg4)-58.0BTB-534 1:4000; anti-Mod(mdg4)-67.2403-610 1:500; anti-Mod(mdg4)-58.0403-534 1:500] were diluted in blocking solution and incubated overnight at 4°. AP- or HRP-conjugated secondary antibodies (Dianova; 1:5000) were added and detection was performed using colorimetric reaction (AP) or ECL system (Amersham Pharmacia Biotech) for HRP-labeled antibodies.
Immunocytochemistry: Ovaries of adult flies were prepared in 3% paratormaldehyde, 0.1% Triton X-100 in PBS and incubated for 2 hr. The ovaries were washed in PBS and dehydrated in 10, 20, 20, 50, 70, 90, and 100% ethanol for 30 min each. The tissue was infiltrated with a mixture of polyethylene glycol (PEG) 1500 and PEG 4000 (2:1). The following steps were used: PEG/ethanol 1:3, 1 hr; PEG/ethanol 1:1, 1 hr; PEG/ethanol 3:1, 1.5 hr; and PEG, 2 hr. Ovaries were embedded and stored at 4°. Sections (2 μm) were performed using a microtome and mounted on a slide coated with 0.1% poly-l-lysine (Sigma, St. Louis). Slides were blocked in 1% BSA in PBS and the anti-Mod(mdg4)-58.0BTB-534 antibody (1:1000 in blocking solution) was added overnight at 4°. The secondary antibody was anti-rabbit-FITC (1:100; Dianova).
Polytene chromosome squashes were prepared according to Silver and Elgin (1978). Glands were prepared in 0.7% NaCl, transferred in squashing solution (45% acetic acid; 1.85% formaldehyde) for 10 min, and squashed. Slides were incubated with primary antibodies [anti-Mod(mdg4)-58.0BTB-534 1:1000; anti-Mod(mdg4)-67.2403-610 1:500; anti-Mod(mdg4)-58.0403-534 1:250] overnight at 4°. The secondary antibodies were FITC or Texas red labeled and used at a dilution of 1:100. DNA staining for sections and squashes was performed with 4′, 6-diamidino-2-phenylindole (DAPI; 1 μg/ml) in PBS for 3–10 min.
RESULTS
mod(mdg4) codes for a large family of alternatively spliced transcripts: The isolation of cDNA clones for mod(mdg4) sharing a common 5′ region but differing in 3′ sequences indicates alternative splicing (Dornet al. 1993b; Gerasimovaet al. 1995; Harveyet al. 1997). In Northern blot analysis, using a genomic 0.5-kb SalI fragment as a probe (indicated in Figure 3), two abundant transcripts of ~2.0 and 2.3 kb have been detected (Figure 1). Sequences of the 0.5-kb SalI fragment are present in all cDNA clones isolated. Both transcripts are found in all developmental stages of Drosophila analyzed, although their abundance is significantly decreased during early larval development (Figure 1). In Western blot analysis with a polyclonal antibody that should recognize all the protein isoforms, at least 12 polypeptides with apparent molecular sizes in the range 70–100 kD are detected in embryonic nuclear extracts (Figure 2A, lane 1). The numerous polypeptides may be explained by complex post-translational protein modifications and/or by the existence of many alternatively spliced transcripts with similar sizes of 2.0 and 2.3 kb. To identify additional putative splice variants, two embryonic cDNA libraries were screened using the 0.5-kb SalI fragment (Figure 1; materials and methods). Altogether, >75 individual cDNA clones have been isolated. These clones were grouped into 21 different cDNA families by restriction analysis and partial sequencing. Fourteen of the different cDNA families are represented by two or more independent clones, whereas 7 are represented by only a single clone, suggesting the existence of even more splice variants generated from the mod(mdg4) locus. With the exception of the cDNA clone mod(mdg4)-58.6, all of them possess poly(A) tails. All cDNAs are generated by alternative splicing using the same acceptor/donor site of exon 4. No alternative splicing was observed within exons 1–4 (cf. Figure 3). We did not identify any cDNA clone matching the sequence of mod(mdg4)1.9 and mod(mdg4)1.8 described by Gerasimova et al. (1995). For each of the 21 different cDNA families, the longest cDNA clone was sequenced on both strands and the putative proteins deduced from the ORFs were named according to the theoretical molecular weight. These sequences are deposited in the EMBL database (for accession numbers see material and methods).
Northern blot analysis of poly(A)+ RNA isolated from indicated stages of Drosophila development. The genomic 0.5-kb SalI fragment (cf. Figure 3) present in all isolated cDNA clones was used as a hybridization probe. In each lane, 5 μg RNA was loaded. Two abundant transcripts in the range 2.0–2.3 kb showing a similar developmental pattern are detected. Note that the transcripts detected represent overlapping signals from different splice variants.
Western blot analysis using antibodies detecting all Mod(mdg4) proteins and domain-specific antibodies. (A) Embryonic nuclear extract (0–12 hr) was fractionated on a 7.5% SDS-PAGE gel. Following transfer to PVDF membrane, two lanes were probed with anti-Mod(mdg4)-58.0BTB-534 antibody detecting all Mod(mdg4) proteins (lane 1) and the domain-specific anti-Mod(mdg4)-67.2403-610 antibody directed against the isoform Mod(mdg4)-67.2403-610 (lane 2). (B) Immunoprecipitation of Mod(mdg4)-58.0 was performed with anti-Mod(mdg4)-58.0403-534-specific antibody using embryonic extracts. The resulting immunoprecipitate was fractionated on a 7.5% SDS-PAGE gel, blotted onto PVDF membrane, and analyzed with anti-Mod(mdg4)-58.0403-534-specific antibody (lane 1) and an affinity-purified anti-Mod(mdg4)-58.0BTB antibody (lane 2) directed against a protein representing almost the BTB domain. The strong signal at 50 kD represents the antibody used for immunoprecipitation.
To show that the two abundant signals obtained in Northern blot analysis are composed of several alternatively spliced transcripts, we used 3′-specific sequences of five different cDNAs as hybridization probes. Specific probes of cDNA clones mod(mdg4)-55.6, mod(mdg4)-56.3, and mod(mdg4)-58.0 detected transcripts with low abundance with a similar size of 2.0 kb. A specific probe derived from cDNA clone mod(mdg4)-67.2 identifies a significantly more abundant transcript with a size of 2.3 kb, whereas the specific cDNA probe from clone mod(mdg4)-55.3 detects a transcript of very low abundance at 2.3 kb (data not shown). The existence of several alternatively spliced transcripts with similar sizes explains the detection of only two abundant transcripts in Northern analysis by probes that include sequences of the common 5′ region.
Genomic organization of the complex mod(mdg4) locus: The molecular structure of the common part of mod(mdg4), the exon/intron structure of two mod(mdg4) cDNAs, and putative regulatory elements identified by sequencing of the corresponding genomic regions are shown in Figure 3. Conserved promoter elements, like two putative CAAT boxes, the TATAA box, a downstream promoter element (DPE), and the transcription start consensus sequence, could be identified at the appropriate distance 5′ from the longest cDNA clones isolated (Figure 3B). With both the TATAA box and the DPE element, mod(mdg4) closely resembles the Drosophila hsp70 gene (Burke and Kadonaga 1997). The common part of all identified cDNA families is encoded by exons 1–4 separated by introns of 242, 77, and 713 bp, respectively. In contrast, larger introns of different sizes separate the common part from the alternatively spliced exons of the two cDNA clones mod(mdg4)-58.0 and mod(mdg4)-55.3, which have been mapped by sequencing the corresponding genomic region shown in Figure 3A. Using the specific 3′ ends of all identified cDNA clones as probes in Southern blot hybridization experiments, five additional specific exons [mod(mdg4)-53.2, mod(mdg4)-55.2, mod(mdg4)-55.6, mod(mdg4)-60.2, and mod(mdg4)-64.2] could be mapped within intron 4 of mod(mdg4)-58.0. According to these results, the specific exons of the earlier described cDNA clones mod-(mdg4)-67.2 [mod(mdg4)2.2] and mod(mdg4)-56.3 (Doom) are localized downstream of exon 5 of cDNA clone mod(mdg4)-55.3. The putative colinear arrangement and the different abundance of alternatively spliced transcripts suggest a complex regulated splicing of a large primary transcript containing the mod(mdg4) exons.
mod(mdg4) encodes a family of related proteins containing a conserved protein consensus motive: All identified cDNA families encode ORFs sharing the common N-terminal part of 402 amino acids but differing in their C termini coding for an additional 28–208 amino acids. The molecular weight of the deduced proteins varies between 46 and 67 kD. The common N terminus contains the BTB/POZ domain, which has been found in >40 different proteins identified in Drosophila, humans, and viruses. Many of these proteins contain zinc finger motifs close to the C terminus (Bardwell and Treisman 1994; Zollmannet al. 1994). No canonical zinc finger motif could be identified in the known Mod-(mdg4) proteins. Sequence comparison of the identified putative Mod(mdg4) proteins revealed a new consensus sequence, which is present in 15 out of 21 Mod(mdg4) proteins (Figure 4A). This consensus sequence consists of 4 hydrophobic amino acids in conserved positions and an additional Cys2His2 motif. The two His residues within the Cys2His2 motif are separated by 1 amino acid, mostly an asparagine. The Cys2His2 motif is represented by the consensus Cys-X6-12-Cys-X17-22--His-X-His (X is any amino acid) indicating variable spacing except the position of the two histidines separated by 1 amino acid, mostly an asparagine. Mod(mdg4) protein isoforms not containing the consensus sequence are shown in Figure 4B.
Molecular map of the mod(mdg4) region and the putative promoter region of mod(mdg4). (A) Genomic region of mod(mdg4) encoding the two alternatively spliced transcripts mod(mdg4)-58.0 and mod(mdg4)-55.3 is shown; ORFs are indicated by solid bars. Below is the restriction map deduced from the two λ phages 129-1 and 129-105; the 7.5-kb BamHI genomic fragment used for rescue experiments is indicated (solid bar). The top part represents a magnification of the region encoding exons 1–4 represented in all identified cDNA families. Insertional mutations identified in this region are indicated by open triangles. Truncated transcripts identified in mutations mod(mdg4)02 and mod(mdg4)neo129 by Northern blot analysis and RT-PCR are shown below the restriction map. Restriction sites indicated are: B, BamHI; H, HindIII; P, PstI; R, EcoRI; and S, SalI. (B) Genomic sequence located at position −1 in the restriction map shown above. Putative CAAT boxes, TATAA box, and DPE element are indicated. The consensus sequence for initiation of transcription (Inr) is underlined.
Harvey et al. (1997) demonstrated the interaction of the specific domain of DOOM, which represents the splice variant Mod(mdg4)-56.3, with the IAP of Orgya pseudotsugata. The presence of the identified consensus sequence in DOOM might indicate its putative function in protein-protein interaction. We could identify a new protein isoform, Mod(mdg4)-54.6, with significant homology to DOOM/Mod(mdg4)-56.3. A sequence comparison of the specific domains of both proteins is shown in Figure 4C. The degree of overall identity is 37% (63% similarity). A functional role of Mod(mdg4)-54.6 in apoptosis has not been shown. Although putative AT-hook motifs in Mod(mdg4)-67.2 and Mod(mdg4)-58.6 and a cluster of acidic amino acids in several proteins have been detected, no other significant homology to known proteins represented in the databases has been identified.
Sequence comparison of the specific C-terminal protein domains of Mod(mdg4) proteins. (A) Alignment of 15 out of 21 Mod(mdg4) proteins containing the consensus sequence. The proteins are named according to their theoretical molecular weight to the left. Note that the common N-terminal part of 402 amino acids containing the BTB domain (cf. Dornet al. 1993a) is not shown. Therefore, all specific protein domains start with amino acid position 403. The alignment (inside the indicated box) was performed by the CLUSTAL X program (Thompsonet al. 1994) with some minor corrections. The consensus sequence (below the indicated box) represents positions where >90% of the aligned proteins have identical amino acids. Because of the varying location of the consensus relative to the C terminus of the different isoforms, the extending C-terminal protein parts are shown below the consensus sequence. (B) Sequence of Mod(mdg4) protein isoforms that do not contain the consensus sequence. Cys and His residues are indicated by boldface letters. Note the presence of at least four Cys or His in all proteins. (C) Comparison of the two protein isoforms Mod(mdg4)-56.3/DOOM and Mod(mdg4)-54.6. Identical amino acid positions are indicated by a colon; chemically similar amino acids are indicated by a point.
Schematic representation of Mod(mdg4) proteins used for generating antibodies. The common antibody anti-Mod(mdg4)-58.0BTB-534 directed against the full-length protein Mod (mdg4) 58.0 should detect all isoforms, whereas the antibodies Mod(mdg4)-58.0403-534 and Mod(mdg4)-67.2403-610 directed against the specific domains of the corresponding protein detect single isoforms.
Mod(mdg4) proteins do not colocalize on polytene chromosomes: Using the antibody anti-Mod(mdg4)-58.0BTB-534 [directed against the full-length Mod(mdg4)-58.0 isoform; cf. Figure 5], a large number of sites on polytene chromosomes were detected (Dornet al. 1993b). Since the common domain was included in the antigen, this antibody should recognize all Mod(mdg4) protein isoforms identified so far. We studied the chromosomal distribution of individual protein isoforms using newly produced specific antibodies generated against fusion proteins containing specific epitopes of two different isoforms, Mod(mdg4)-67.2 (aa 403–610) and Mod(mdg4)-58.0 (aa 403–534; Figure 5). Mod-(mdg4)-67.2 is encoded by an abundant 2.3-kb transcript, whereas cDNA clone mod(mdg4)-58.0 represents a significantly less abundant 2.0-kb transcript.
The anti-Mod(mdg4)-58.0BTB-534 antibody detects at least 12 polypeptides in embryonic nuclear extracts (Figure 2A, lane 1), whereas the specific antibody anti-Mod(mdg4)-67.2403-610 detects 2 polypeptides at molecular weights of 93 and 99 kD (Figure 2A, lane 2), although a single polypeptide of 67 kD was expected. Both the existence of 2 cross-reacting polypeptides and their increased apparent molecular size in SDS-PAGE gels suggest post-translational modifications. This is supported by the presence of several putative phosphorylation sites within the common part of the N-terminal 402 amino acids. Comparison of the theoretical molecular weight of the identified Mod(mdg4) proteins in the range of 46 to 67 kD and the apparent molecular weight of polypeptides detected in Western blot analysis (70–100 kD) suggests a general shift in electrophoretic mobility of ~25 kD. The second specific antibody, anti-Mod(mdg4)-58.0403-534, did not detect a polypeptide in Western blot of embryo nuclear extracts. However, after immunoprecipitation by the anti-Mod(mdg4)-58.0403-534 antiserum using embryonic extracts, 2 polypeptides at 75 kD are clearly detected by this antibody (Figure 2B, lane 1). The 2 polypeptides also react with the affinity-purified anti-BTB antiserum (Figure 2B, lane 2). No cross-reaction of the immunoprecipitate with the specific anti-Mod(mdg4)-67.2403-610 antibody was found, indicating the selectivity of both specific antisera.
Localization of Mod(mdg4) proteins and the specific distribution of the two protein isoforms Mod(mdg4)-58.0 and Mod(mdg4)-67.2 along polytene chromosomes. Double staining of wild-type chromosomes using affinity-purified anti-Mod(mdg4)-58.0BTB-534 antibody (A) and anti-Mod(mdg4)-67.2403-610 (B). Merged images (C) indicate the overlapping sites (yellow). Staining of polytene chromosomes using antibodies anti-Mod(mdg4)-58.0BTB-534 (D) and anti-Mod(mdg4)-58.0403-534 detecting the isoform Mod(mdg4)-58.0 (E). Overlapping signals (yellow) are shown in the merged image (F). Costaining of polytene chromosomes using the two domain-specific antibodies anti-Mod(mdg4)-58.0403-534 (G) and anti-Mod(mdg4)-67.2403-610 (H). Double exposure (I) demonstrates the partial (yellow) but not complete overlap of both Mod(mdg4) protein isoforms. Mod(mdg4)-58.0-specific signals are indicated by long arrows, and putative overlapping signals of Mod(mdg4)-58.0 and Mod(mdg4)-67.2 are indicated by short arrows.
The chromosomal distribution of the Mod(mdg4)-67.2 protein isoform was studied by double staining of polytene chromosomes with the common antibody anti-Mod(mdg4)-58.0BTB-534 and the specific antibody anti-Mod(mdg4)-67.2403-610. Both antibodies detect a large number of sites (Figure 6, A and B). The merged images (Figure 6C) show an overlap at most (yellow signals) but not all sites. A small number of sites, which are exclusively detected by the anti-Mod(mdg4)-58.0BTB-534 antibody (green signals), correspond to binding sites of Mod(mdg4) proteins other than Mod(mdg4)-67.2. These are located at the telomeres of the autosomes and the X chromosome and a number of loci like 2C, 7D, and 95D (labeled by arrows in Figure 6C). In contrast, the other specific antibody anti-Mod(mdg4)-58.0403-534 detects only ~50 sites on polytene chromosomes (Figure 6, E and G Table 1). A total of 24 sites have been found in all preparations analyzed so far, with 3 sites standing out by a very prominent staining. Of the sites listed in Table 1, 23 stain more weakly and/or were not detected in all nuclei analyzed.
Costaining of polytene chromosomes, using anti-Mod(mdg4)-58.0BTB-534 and the specific anti-Mod(mdg4)-58.0403-534 antibody, results in a complete overlap of sites detected by the specific antibody (yellow signals, Figure 6F). However, the majority of sites are recognized exclusively by the antibody detecting all Mod(mdg4) proteins (red signals). This result indicates that Mod(mdg4)-58.0 is present in a small subset of Mod(mdg4) binding sites. Next, we compared the distribution of the two protein isoforms Mod(mdg4)-58.0 and Mod(mdg4)-67.2 by costaining with the two specific antibodies (Figure 6, G and H, respectively). In merged images only, a subset of sites is recognized by both antibodies (Figure 6I, yellow signals). However, at many sites both proteins exclude each other, clearly indicating a differential distribution of the two protein isoforms Mod(mdg4)-58.0 and Mod(mdg4)-67.2.
Binding sites of Mod(mdg4)-58.0 on polytene chromosomes
Isolation and genetic analysis of mod(mdg4) mutations: First mutations of mod(mdg4) have been identified by their modifying effect on several gypsy-induced mutations (Georgiev and Gerasimova 1989). The independently isolated P-transposon-induced insertional enhancer of position effect variegation E(var)3-93Dneo129 was used for molecularly defining the locus (Dornet al. 1993b). Molecular cloning of the mod(mdg4) locus revealed that both E(var)3-93D and mod(mdg4) are identical genes (Gerasimovaet al. 1995; Gerasimova and Corces 1998). These studies also proved that the previously isolated mod(mdg4) mutations show a dominant enhancer effect on PEV. It was concluded that mod-(mdg4) behaves as a typical enhancer of position effect variegation, which connects insulator function with changes in chromatin structure.
Mutations of mod(mdg4) are characterized not only by their dominant enhancer effect on PEV, but also by their result in lethality or semilethality and female sterility (Dornet al. 1993b; Gerasimovaet al. 1995). To identify new alleles of mod(mdg4), we tested >100 independently isolated dominant enhancers of PEV mutations (Dornet al. 1993a) for viability and female fertility in trans-heterozygotes with mod(mdg4)neo129 and Df(3R)GC14. In these studies, four new mod(mdg4) alleles were identified. Two mutations, mod(mdg4)03 and mod-(mdg4)05, were induced by P-element mutagenesis and two, mod(mdg4)02 and mod(mdg4)04, were spontaneous in origin (Table 2).
Deficiency Df(3R)GC14 uncovers mod(mdg4) and several lethal complementation groups (Azpiazu and Frasch 1993). Two of the mutations isolated in a screen for EMS-induced recessive lethals uncovered by Df(3R)GC14 (Azpiazu and Frasch 1993) proved to be allelic to mutations mod(mdg4)06 and mod(mdg4)07 (Table 2). The P(w+)142 transposon-induced mutation affects the same complementation group, and several deficiencies have been generated by imprecise excision of the transposon (Azpiazu and Frasch 1993). All these deficiencies, including P142Δ32, delete sequences in the common part of the mod(mdg4) locus and display a strong dominant enhancer effect on PEV (Table 2; data not shown).
The mutations differ in the strength of their dominant enhancer effect on white variegation in wm4 and can be arranged in an allelic series. The strongest enhancer effect is shown by mutations mod(mdg4)R32, mod(mdg4)04, mod(mdg4)06, mod(mdg4)07, and deletion P142Δ32. Weaker enhancer effects are found in genotypes carrying mod(mdg4)neo129, mod(mdg4)02, and mod-(mdg4)03, whereas mutation mod(mdg4)05 shows only a weak enhancer effect (Table 2). Enhancement of position effect variegation has been quantified by red eye pigment measurements in wm4 flies also carrying the strongly dominant suppressor of position effect variegation Su(var)2-101.
Complementation analysis between the newly identified mod(mdg4) mutations and mod(mdg4)neo129 revealed a rather complex complementation pattern (Table 3). The complementation analysis showed that two mutations, mod(mdg4)05 and mod(mdg4)03, complement or partially complement most of the other alleles (Table 3). Allele mod(mdg4)05 is viable with all mutations, including Df(3R)GC14. Trans-heterozygous females are fertile, with the exception of mod(mdg4)05/mod(mdg4)03 females (Table 3). mod(mdg4)03 partially complements most alleles, and all trans-heterozygotes tested are female sterile. These results suggest a weak hypomorphic nature of mutations mod(mdg4)05 and mod(mdg4)03. Both mutations belong to the group of alleles that display an intermediate or weak enhancer effect.
In an excision analysis of the insertional mutation mod(mdg4)neo129, the recessive lethal allele mod(mdg4)R32 was isolated by screening for ry−-associated phenotype and is lethal with all mod(mdg4) alleles except mod(mdg4)05 and mod(mdg4)03. An almost identical complementation pattern is found for alleles mod(mdg4)04, mod(mdg4)06, and mod(mdg4)07. All of these alleles show the strongest enhancer effect and are suggested to represent amorphic or strongly hypomorphic mutations in respect to dominant enhancement of PEV and recessive lethality.
PEV enhancer effect and homeotic effects of mod(mdg4) mutations
Mutations of mod(mdg4) were furthermore shown to display dominant homeotic effects characteristic for trx group genes in adult flies (Dornet al. 1993b; Gerasimova and Corces 1998). All of the newly identified alleles show significant homeotic effects (Table 2). In males trans-heterozygous for Df(3R)redtrx and the mutations mod(mdg4)neo129, mod(mdg4)R32, mod(mdg4)03, mod(mdg4)04, or mod(mdg4)05, homeotic transformation of abdominal segment A5 to A4 is strongly increased compared to trans-heterozygotes with mod(mdg4)02, mod-(mdg4)06, or mod(mdg4)07. However, no direct correlation between the strength of the PEV enhancer effect and the frequency of homeotic transformation is found. Considering the molecular complexity of the locus, the mutations might differentially affect various isoforms of the mod(mdg4) locus.
Complementation analysis of mod(mdg4) alleles
Interaction of mod(mdg4)neo129 and mutations in other trx group genes in homeotic transformation
Mutations in mod(mdg4) and other trx group genes additively interact in homeotic transformation as well as enhancement of PEV: To test for interaction between mod(mdg4)neo129 and mutations in trx group genes in their effect on homeotic transformation and dominant enhancement of PEV, we constructed several cis combinations between mod(mdg4)neo129 and mutations of other trx group genes, including brahma, vertandi, trithorax, and E(var)3-4. Phenotypic effects on PEV were quantified in wm4 flies also carrying the strong dominant PEV supprespsor mutation Su(var)2-101 by red eye pigment measurements. Homeotic transformation of abdominal segments A5 to A4 was quantified by arbitrary units (materials and methods).
Strong additive effects on homeotic transformation of A5 to A4 were found in Df(3R)redtrx mod(mdg4)neo129/+ males (Table 4). In these males, about one-third of the abdominal segment A5 was transformed to A4. Df(3R)redtrx mod(mdg4)neo129/bx34e flies showed strong transformation of T2 to T3 in both females and males (data not shown).
No further enhancement of homeotic transformation was found in Df(3R)redtrx mod(mdg4)neo129/brm2 males (Table 4). The two dominant enhancer mutations, E(var)3-401 and mod(mdg4)neo129, alone cause only weak homeotic transformation. A significant increase of A5 to A4 transformation can be observed in the double heterozygous E(var)3-401 mod(mdg4)neo129/+ males (Table 4).
Although none of the trx alleles nor Df(3R)redtrx affects PEV, strong enhancement of white variegation was found in wm4; Df(3R)redtrx mod(mdg4)neo129/+ flies. Elevated enhancer effects on PEV were also detected in vtd2 mod (mdg4)neo129/+ males, whereas brm2 mod(mdg4)neo129/+ males were not significantly different compared to mod (mdg4)neo129/+ controls (Table 5). Our results show that a deletion of trx as well as a mutation in vtd strongly elevate the enhancer effect of mod(mdg4)neo129 on position effect variegation in wm4. Therefore, these mutations not only show additive effects on homeotic transformation but also in their effect on position effect variegation. Different alleles of brahma and vertandi including brm2 and vtd2 show only weak enhancer effects on PEV (Table 5). Additive effects on enhancement of PEV were found between brm and trx in wm4/Y; brm2 Df(3R)redtrx/+ males and enhancement of PEV is about three times stronger compared to wm4/Y; brm2/+ males. No significant difference in enhancement of PEV was observed in combinations between E(var)3-401 and mod(mdg4)neo129, which may be attributed to the strong enhancer effect of the E(var)3-401 mutation.
Interaction of mod(mdg4)neo129 and mutations in other trx group genes in enhancement of PEV
Our data indicate that mod(mdg4) not only interacts with trx group genes in transcriptional regulation of homeotic genes but also in gene silencing caused by PEV. In earlier studies of mod(mdg4)neo129, we reported paternal imprinting-like enhancer effects (Dornet al. 1993b). Male offspring produced by mod(mdg4)neo129/+ heterozygous fathers show PEV enhancement independent of whether they are mod(mdg4)neo129/+ or +/+ in genotype. These effects can be observed for all other mod(mdg4) mutant alleles (Table 2). Strongly elevated paternal enhancer effects are found in offspring males produced by crosses with fathers of cis-heterozygotes of mod(mdg4)neo129 and the tested mutations of trx group genes (Table 5). The two cis combinations, Df(3R)redtrx mod(mdg4)neo129 and vtd2 mod(mdg4)neo129, show the strongest paternal imprinting-like enhancer effect. Interestingly, a significant paternal enhancer effect is also detected in crosses with brm2 Df(3R)redtrx/+ fathers. These results suggest a functional interaction of several trx group genes not only in the control of homeotic gene expression but also in gene silencing caused by a change in higher order chromatin organization in PEV.
Antibody staining of thin-sectioned stage 10 egg chambers from Drosophila wild-type ovaries (A) and ovaries from homozygous mod-(mdg4)neo129 females (C) using the anti-Mod(mdg4)-58.0BTB-534 antibody detecting all Mod-(mdg4) proteins. Note the significantly reduced staining in all nuclei of mutant egg chambers. Images shown in B and D represent DNA staining using DAPI of the same egg chambers shown in A and B, respectively.
Molecular analysis of mod(mdg4) mutations: Because of the molecular complexity of mod(mdg4), it is important to know the molecular nature of mod(mdg4) mutant alleles. Mutations involved in sequences encoding the common exons 1–4 should affect all identified protein isoforms. Five of the nine mod(mdg4) mutant alleles are transposon induced. The mod(mdg4)neo129 mutation was induced by insertion of the pUChsneory+ transposon 121 bp downstream from the 3′ junction of the third exon. Northern blot analysis using poly(A)+ RNA isolated from heterozygous flies revealed a truncated transcript of ~1.0 kb slightly less abundant compared to the wild-type transcripts (data not shown). For a second P-induced allele, mod(mdg4)03, restriction fragment length polymorphism within the genomic region encoding the common part of all transcripts was identified by Southern blot analysis. Isolation and sequencing of appropriate recombinant phages from a genomic library constructed from this mutant revealed the insertion of a truncated P-element 120 bp downstream from the putative TATAA box within the 5′ untranslated region (UTR). However, in Northern blot analysis of poly(A)+ RNA isolated from heterozygous mod(mdg4)03 adults, no aberrant transcript could be detected. The insertion of the w+ transposon in mutation P(w+)142 could be located 40 bp downstream from the TATAA box in the 5′ UTR (M. Frasch, personal communication). Both mutations might result in reduced amounts of mod(mdg4) transcripts. In flies carrying the allele mod(mdg4)02, which was obtained as a spontaneous mutation in a hybrid dysgenic cross, an abundant truncated transcript of ~1.2 kb was detected in Northern blot analysis (data not shown). Cloning and sequencing of the mutant allele revealed the insertion of a gypsy-like retrotransposon within the third intron, 84 bp downstream from the exon/intron junction of exon 3. All mutations represent independent insertions of transposable elements within a region of ~1 kb of the mod(mdg4) locus, indicating a hot spot for insertional mutations.
The truncated transcripts detected in mutations mod-(mdg4)neo129 and mod(mdg4)02 are expected to result from aberrant splicing. Sequencing of DNA fragments obtained by RT-PCR revealed that the aberrant transcripts contain transposon sequences. Putative polyadenylation signals within the transposons are used to produce the detected truncated transcripts (Figure 3A). However, the coding capacity of both truncated transcripts is too small to encode functional Mod(mdg4) proteins.
Mod(mdg4) proteins are essential in oogenesis and early embryogenesis: The mutant allele mod(mdg4)neo129 shows temperature-sensitive pupal lethality. Homozygous females, obtained at 29°, produce a small number of eggs that do not show signs of further development. Ovaries of these homozygous females are of variable size, and ovarioles are frequently reduced in number and contain fewer egg chambers. In whole-mount in situ hybridization, large amounts of mod(mdg4) transcripts could be detected in nurse cells of wild-type egg chambers (data not shown). To determine if the female sterility of homozygous mod(mdg4)neo129 females is due to the reduced amount of Mod(mdg4) proteins during oogenesis, we compared the distribution of Mod(mdg4) proteins in thin sections of wild-type and mutant vitellogenic egg chambers. In immunocytological analysis, using the anti-Mod(mdg4)-58.0BTB-534 antibody, we found Mod(mdg4) proteins in follicle and nurse cell nuclei of wild-type stage 10 egg chamber (Figure 7A). In contrast, we found only very weak staining in egg chambers of homozygous mod(mdg4)neo129 females (Figure 7C). In nurse cell nuclei, only very little staining can be observed, whereas the nuclei of follicle cells, which surround the oocyte at this stage, do not stain at all. Additionally, in wild-type oocytes, we identified Mod(mdg4) proteins in the germinal vesicle, whereas no protein can be detected in the germinal vesicle of mutant homozygotes (Figure 7, A and C). In whole-mount in situ hybridization analysis using the full-length cDNA clone mod(mdg4)-58.0 as a hybridization probe, we also found strongly reduced levels of mod(mdg4) transcripts in homozygous mutant egg chambers (data not shown). The reduced amount of Mod(mdg4) proteins in mutant egg chambers correlates with the observed maternal effect lethality, indicating an essential function of Mod(mdg4) proteins in oogenesis and early embryonic development.
Reduced levels of Mod(mdg4) proteins in larval stages of homozygous mod(mdg4) mutants. (A) Staining of polytene chromosomes of homozygous mod(mdg4)02 larvae using the anti-Mod(mdg4)-58.0BTB-534 antibody, which detects all Mod(mdg4) protein isoforms. (B) Staining of the same set of chromosomes with anti-histone H1 antibody. (C) Fluorescent micrograph of polytene chromosomes from homozygous mod(mdg4)neo129 larvae stained with anti-Mod(mdg4) 58.0BTB-534 antibody. Note the significantly reduced intensity of signals.
Late larval/early pupal lethality of mod(mdg4) mutants is due to reduced levels of Mod(mdg4) proteins: The mutant alleles mod(mdg4)02 and mod(mdg4)neo129 show late larval/early pupal lethality. Immunocytological analysis of polytene chromosomes from homozygous third instar larvae should prove the correlation between lethality and reduced levels of Mod(mdg4) proteins at this developmental stage. Using the antibody anti-Mod(mdg4)-58.0BTB-534, little or no protein can be detected by immunostaining of polytene chromosomes from homozygous mod(mdg4)02 larvae (Figure 8A). The same result was obtained with a specific antibody directed against the Mod(mdg4)-67.2 protein (data not shown), whereas the costaining with an anti-histone H1 antibody as a control does not differ significantly from staining of wild-type chromosomes (Figure 8B). Salivary gland polytene chromosomes from homozygous mod (mdg4)neo129 third instar larvae showed significantly reduced staining with the anti-Mod(mdg4)-58.0BTB-534 antibody (Figure 8C). A plausible hypothesis to explain the differences in immunostaining of polytene chromosomes from the two insertional mutants could be the strength of transcription termination within the different transposons, allowing the production of functional transcripts at low frequency in the case of the pUChs-neory+ transposon in mod(mdg4)neo129 mutation but not (or only very rarely) in mod(mdg4)02 homozygotes containing the gypsy-like insertion. This hypothesis could also explain the observed semilethality of mod(mdg4)neo129 at 29°, assuming an increased accumulation of correctly spliced mod(mdg4) transcripts during development at elevated temperature. We suppose that insufficient amounts of functional Mod(mdg4) proteins result in developmental arrest.
mod(mdg4) mutant phenotypes can be partially rescued by a genomic fragment encoding the common part of mod(mdg4) transcripts: The common N terminus of 402 amino acids, shared by all identified Mod(mdg4) proteins, contains a Glu/Thr-rich domain in addition to the BTB/POZ domain (Dornet al. 1993b; cf. Figure 5). To study whether this common N-terminal region contains intrinsic functions, the 7.5-kb genomic BamHI fragment (indicated in Figure 3A) containing the coding region for the common part of 402 amino acids and the identified conserved promoter elements was used for P-element-mediated transformation. Several second chromosomal transgenic lines containing this genomic fragment could be established (materials and methods). Surprisingly, the lethality of mod(mdg4)neo129 and mod(mdg4)02 mutant homozygotes and several trans-heterozygotes containing other mod(mdg4) alleles could be partially rescued in the presence of the genomic 7.5-kb BamHI fragment (Table 6). However, no rescue was found for EMS-induced mutations mod(mdg4)06 and mod(mdg4)07.
Partial rescue of recessive lethality of mod(mdg4) alleles in the presence of the transgene P (w+7.5kb BamHI)
To determine whether homozygous mod(mdg4)neo129 mutants containing the transgene were sterile, we examined eggs deposited from these females after mating with wild-type males. Development of eggs is arrested at different stages of embryogenesis, although occasional first larval instar escapers were found. This result indicates a partial rescue of the maternal effect lethal phenotype.
Significant homeotic transformation of abdominal segment A5 to A4 was found in P(w+7.5kb BamHI); mod(mdg4)02/mod(mdg4)02 males and P(w+7.5kb BamHI); mod(mdg4)neo129/mod(mdg4)neo129 males (Figure 9, A and B), indicating that the transgene does not rescue this mutant phenotype. Elevated additive effects of homeotic transformation in Df(3R)redtrx mod(mdg4)neo129/mod-(mdg4)neo129 males are also not affected by the P(w+7.5kb BamHI) transgene (Figure 9C). Two conclusions can be drawn from these results. First, the presence of a transgene containing sequences encoding the common part of mod(mdg4) but lacking the alternatively spliced exons is able to partially rescue recessive lethality. Second, the partial rescue of recessive lethality results in females with maternal effect lethality and males with significant homeotic transformation, indicating that sequences encoding the specific domains and/or their expression at sufficient levels are important for wild-type function of Mod(mdg4) proteins.
DISCUSSION
Mod(mdg4) protein isoforms are differentially distributed on polytene chromosomes: Our molecular analysis revealed that mod(mdg4) produces a large number of transcripts by alternative splicing. We have identified 17 new splice variants. All deduced proteins contain a common N terminus of 402 amino acids encompassing the most N-terminal BTB/POZ domain and different C termini. Our results clearly indicate a differential distribution of at least two Mod(mdg4) proteins, Mod(mdg4)-58.0 and Mod(mdg4)-67.2, along polytene chromosomes. Whereas Mod(mdg4)-67.2 is found at the majority of sites, labeled by the antibody anti-Mod(mdg4)-58.0BTB-534 detecting all protein isoforms, the other isoform is restricted to a small subset of sites. The binding of Mod(mdg4)-58.0 and Mod(mdg4)-67.2 at different sites suggests that at least these two Mod(mdg4) isoforms participate in transcriptional regulation of different sets of genes. We suppose that the specific C-terminal domains play a critical role in directing the isoforms to different binding sites, possibly through specific interactions with other proteins. Two other observations are consistent with this hypothesis. Gerasimova et al. (1995) demonstrated by genetic and in vitro binding assays an interaction of Mod(mdg4)-67.2 [Mod(mdg4)2.2] with Su(Hw), a zinc finger protein that binds to gypsy sequences. Both proteins are implicated in the function of chromatin insulator sequences present in the gypsy transposon. Using the yeast two-hybrid system, Harvey et al. (1997) identified one of the Mod(mdg4) isoforms, DOOM [Mod(mdg4)-56.3], by its ability to interact with the baculovirus inhibitor of apoptosis protein. In coimmunoprecipitation experiments, this interaction could be clearly localized to the specific C-terminal domain of DOOM. Together these results suggest that the large number of protein isoforms generated from mod(mdg4) reflects the functional diversity of individual Mod(mdg4) proteins. The GAGA factor, encoded by the Trl gene, was shown to be involved in nucleosome remodeling in regulatory regions of many genes (reviewed in Wilkins and Lis 1997). Mutations in Trl and mod(mdg4) display very similar genetic properties, e.g., enhancement of PEV, paternal effects, and homeotic transformation. The generation of different GAGA isoforms containing a common N terminus of 377 amino acids with an N-terminal BTB/POZ domain has been demonstrated. However, in contrast to mod(mdg4), a colocalization of two different GAGA isoforms on polytene chromosomes and their ability to form heterodimers could be demonstrated by coimmunoprecipitation (Benyajatiet al. 1997).
Homeotic transformation of abdominal segment A5 to A4 in homozygous mod(mdg4) mutant males containing the 7.2-kb BamHI transgene. (A) P(w+7.5kbBamHI)/+; mod(mdg4)02/mod(mdg4)02 males, (B) P(w+7.5kbBamHI)/+; mod(mdg4)neo129/mod(mdg4)neo129 males, and (C) P(w+7.5kb BamHI)/+; Df(3R)redtrx mod(mdg4)neo129/mod(mdg4)neo129 males.
That the protein consensus sequence contains a Cys2His2 motif within the specific protein domains of most Mod(mdg4) isoforms may be of functional importance. In contrast to canonical zinc-finger motifs of the Cys2His2 type, the one found here has distinct features. The two histidine residues are separated by only one amino acid residue, and the consensus sequence extends N-terminal with additional conserved aromatic amino acid positions. The presence of the conserved sequence in the specific protein domain of DOOM implicates its putative involvement in protein-protein interaction with IAP. Disruption of this interaction by mutagenesis of the highly conserved amino acid positions could test this hypothesis. However, five of the different isoforms do not contain the identified consensus sequence, including Mod(mdg4)-58.0 and Mod(mdg4)-67.2. The functional significance of the presence of several Cys and His residues in these isoforms remains unknown.
Pleiotropic effects of mod(mdg4) mutations: Genetic analysis of several mod(mdg4) mutant alleles revealed pleiotropic effects. All mutations are dominant enhancers of PEV and display paternal enhancer effects. Additionally, mod(mdg4) mutations have been demonstrated to display properties typical for trx group genes (Dornet al. 1993b; Gerasimova and Corces 1998). Enhanced homeotic transformation has been observed in trans and cis combinations with several mutations in other trx group genes, suggesting a possible interaction of the corresponding proteins (Gerasimova and Corces 1998; this work). This is supported by the observed interactions in PEV enhancement. Double heterozygous Df(3R)redtrx mod(mdg4)neo129/+ + males strongly enhance PEV in wm4 flies, whereas the trx mutation itself has no effect. In our earlier study, we described paternal enhancer effects (Dornet al. 1993b). Furthermore, the finding of strong paternal enhancer effects in +/+ male offspring derived from Df(3R)redtrx mod(mdg4)neo129/+ + fathers also implies an interaction of the corresponding proteins in gene silencing in PEV. Based on the finding of different distribution of Trx and Mod(mdg4) proteins in diploid interphase nuclei and the altered distribution of Mod(mdg4) proteins in the background of trx mutations, Gerasimova and Corces (1998) proposed a two-tier model for chromatin assembly. According to this model, the formation of complexes containing Trx precedes the assembly of Mod(mdg4) proteins.
Most of the molecularly characterized mod(mdg4) mutations involve sequences within the common 5′ region. These mutations would be expected to affect all Mod(mdg4) protein isoforms, explaining the observed pleiotropic mutant effects. Although we have demonstrated the differential distribution of two protein isoforms, we do not know if the loss of single isoforms causes distinct mutant phenotypes. This would be expected if Mod(mdg4) proteins have specific functions in chromatin. Mutations within the specific protein domains of different isoforms should allow a further functional dissection of mod(mdg4).
mod(mdg4) is expressed at high levels during oogenesis: The presence of large amounts of Mod(mdg4) proteins in all stages of oogenesis and early embryogenesis indicates a strong maternal component (this work; K. Büchner and R. Dorn, unpublished results). The significantly reduced amounts of Mod(mdg4) proteins detected in egg chambers of homozygous mod(mdg4)neo129 females and the failure of eggs to foster further development indicate important functions of mod(mdg4) during oogenesis and early embryonic development. The presence of Mod(mdg4) in both nurse cell and follicle cell nuclei and the supposed role as a general transcriptional regulator suggest that mod(mdg4) is required for control of maternal genes during oogenesis. This is in agreement with the supposed role of Mod(mdg4) protein(s) in mediating the function of chromatin insulator sequences as a prerequisite for correct promoter-enhancer interactions (Gerasimova and Corces 1998). In the embryo, Mod(mdg4) proteins do not become localized to the nuclei until cleavage cycle 9 (K. Büchner and R. Dorn, unpublished results), further arguing against a function in chromatin organization during early cleavage cycles.
We have shown that a transgene containing the common part of Mod(mdg4) can partially rescue the recessive lethality of mod(mdg4) mutant alleles. This result can be explained by the expression of a truncated protein containing the 402-amino-acid common N-terminal region and the ability to partially replace the function of full-length Mod(mdg4) proteins. However, we were not able to detect a protein of the expected molecular size in the transgenic animals, which may be due to the limited sensitivity of Western blot analysis. Expression of a tagged protein under control of the hsp70 promoter from a transgene will be required to prove the proposed function of the common N-terminal peptide.
Acknowledgments
We are grateful to Dr. M. Frasch for providing mutations AI117 and AI351 [corresponding to mod(mdg4)06 and mod(mdg4)07, respectively] and P(w+)142 and revertants of this insertional mutation, including P142Δ32. The anti-histone H1 antibody was kindly provided by Dr. U. Grossbach. We would like to thank M. Reich for isolation of revertants from mod(mdg4)neo, G. Hause for advice in immunocytological analysis of ovaries, and M. Kube and C. Frost for technical assistance. We thank V. Pirrotta for critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft to R.D. (Do407/1-2 and 1-3).
Footnotes
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Communicating editor: T. C. Kaufman
- Received July 27, 1999.
- Accepted January 12, 2000.
- Copyright © 2000 by the Genetics Society of America
