Regena (Rga), a Drosophila Homolog of the Global Negative Transcriptional Regulator CDC36 (NOT2) from Yeast, Modifies Gene Expression and Suppresses Position Effect Variegation

Corresponding author: James A. Birchler, 117 Tucker Hall, University of Missouri-Columbia, Columbia MO 65211, birchler{at}biosci.mbp.missouri.edu (E-mail).

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A mutation in Regena (Rga) was isolated in screens for modifiers of white eye color gene expression. The reduction in the level of the Rga product results in a complex modulation of white mRNA both positively and negatively, depending on the developmental stage. In addition to white, Rga also affects the expression of several other tested genes, with one of them, Vinculin, being regulated in a strong sex-specific manner. Rga was cloned by transposon tagging. Its predicted product lacks any recognized nucleic acid–binding motif but is homologous to a global negative transcriptional regulator, CDC36 (NOT2), from yeast. Rga also acts as a suppressor of position effect variegation, suggesting that a possible function of Rga could be mediation of an interaction between chromatin proteins and the transcriptional complex.

TRANSCRIPTIONAL initiation is controlled by the presence and activity of specific nuclear factors that directly bind to the short sequence motifs in the promoter regions of genes, thus, interacting with components of the general transcription machinery. Regulation can also be affected by intermediary proteins that govern transcription indirectly by association with the promoter via protein-protein interactions. Their function is to mediate the interaction of transcription factors with the RNA polymerase II complex or to establish or maintain the proper chromatin structure. For a particular gene, a unique combination of all available regulatory proteins and cis-acting elements determines its specific expression pattern in development.

The thorough studies of intermediary proteins in yeast resulted in identification and molecular characterization of a set of global positive and negative regulators affecting the expression of numerous genes (for review, see STRUHL 1993 ). Based on genetic and biochemical analyses, it was proposed that some of them act as heterologous multiprotein complexes. For example, Cdc39p (Not1p), Cdc36p (Not2p), Not3p, and Mot2p (Not4p) presumably form a 500-kD complex that inhibits transcription by affecting the ability of transcription factor TFIID for TATA element utilization (COLLART and STRUHL 1994 ). Another potential complex is formed by Spt4p, Spt5p, and Spt6p, which negatively regulate transcription of several loci. Unlike Not proteins, these were hypothesized to repress gene expression by affecting chromatin structure (SWANSON and WINSTON 1992 ) analogous to that postulated for Polycomb group proteins (PARO 1990 ). One such component, Spt6p, controls chromatin structure by direct interactions with histones H3 and H4 (BORTVIN and WINSTON 1996 ).

The white locus provides a useful model system in Drosophila where such interactions can be studied. A large number of genes that modify white expression have been identified (BINGHAM and ZACHAR 1985 ; CHAPMAN and BINGHAM 1985 ; RABINOW et al. 1991 ; BIRCHLER et al. 1994 ; BHADRA and BIRCHLER 1996 ; BHADRA et al. 1997A , BHADRA et al. 1997B ). Because such modifiers usually produce a dosage effect, they were proposed to be examples of genes that are the underlying basis of aneuploid syndromes (BIRCHLER and NEWTON 1981 ) and various types of dosage compensation (DEVLIN et al. 1982 ; BIRCHLER et al. 1989 , BIRCHLER et al. 1990 ; BIRCHLER 1996 ; BHADRA et al. 1997A , BHADRA et al. 1997B ). The zeste gene, the only previously cloned modifier of white, encodes a protein that specifically binds to transcriptional enhancers in the vicinity of the promoter region of the white gene and controls long-distance interaction in chromosomes (BENSON and PIRROTTA 1987 ; for review, see PIRROTTA 1990 ). The zeste product shows extensive self-assembly properties and can form multimeric complexes connecting distantly located regions of DNA to one another (BICKEL and PIRROTTA 1990 ). Several other modifiers have been found to interact with the white gene. They produce both direct correlative and inverse dosage effects on white expression. For example, mutant alleles of the Weakener-of-white (Wow) locus increase the level of the white mRNA in larvae but decrease it in adults (BIRCHLER et al. 1994 ). Modifier of white (Mow) was shown to have a differential effect on gene expression in males and females (BHADRA and BIRCHLER 1996 ). Inverse regulator-a (Inr-a) modifies the white gene only in larval and pupal stages, when the majority of pigment is deposited (RABINOW et al. 1991 ). In addition to white, two other tested genes, brown and scarlet, are also affected by these modifiers. Moreover, Wow and Mow were recognized as weak suppressors of position effect variegation (PEV; BIRCHLER et al. 1994 ; BHADRA and BIRCHLER 1996 ), which implies a connection between regulation of higher order chromatin structure and modulation of gene activity.

Using white, we are interested in isolating as complete a set of regulators as possible for a single target locus to understand how the numerous modifiers affect gene expression. In this report, a newly identified Drosophila gene, Regena (Rga), which modulates the expression of the white locus, is characterized. The product of Rga is essential for normal Drosophila viability, as evidenced by the fact that the mutant allele induced by a P-element insertion is semilethal. Rga was found to act as a suppressor of PEV and regulate the expression of several unrelated genes. Because it affects expression of numerous genes, it was named Regena (Rga) (Regulator of gene activity). The predicted protein product contains a domain homologous to the yeast general negative transcription regulator CDC36 (NOT2).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly stocks:
Flies were raised on standard Drosophila media at 25°. Genetic markers used here can be found in LINDSLEY and ZIMM 1992 .

l(3)03834, a single PZ element (MLODZIK and HIROMI 1992 ) insertion on chromosome 3 (83B4-5), was identified in a large screen for autosomal mutations affecting the eye color of white-apricot flies. To determine whether this insertion is responsible for the mutant phenotype, the P element was mobilized by crossing l(3)03834/TM3, Sb flies to the delta 2-3, Sb/TM6, Ubx strain (ROBERTSON et al. 1988 ). The F₁ l(3)03834/delta 2-3, Sb males were crossed to the balancer stock TM3, Ser/MKRS individually. The Sb, non-Ser progeny (l(3)03834/MKRS) were screened for rosy^- flies, which were mated to TM3, Ser/MKRS to establish a stock.

For the developmental Northern analysis, genetic crosses were performed essentially as described (BIRCHLER et al. 1994 ). Briefly, l(3)03834/TM3, Sb females were crossed to T(2;3)CyO, Cy Tb ch translocation males. The F₁ males containing this translocation heterozygous with l(3)03834 were mated to Canton S females. The Tb marker allows discrimination between +/+ and l(3)03834/+ classes at the larval and pupal stages, while the Curly marker allows this distinction in adults.

DNA manipulation and cDNA libraries:
All standard DNA manipulations were performed as described in SAMBROOK et al. 1989 .

Genomic DNA was isolated from 50 flies by standard procedures with some modifications (ALATORTSEV 1988 ). The fragment flanking the insertion site in l(3)03834 was rescued as described in KARPEN and SPRADLING 1992 . The enzymes XbaI and NheI were used for double digestion of 1 µg of the genomic DNA isolated from flies carrying the P element insertion. After inactivation and dilution, a ligation reaction was performed in 200 µl at 16° overnight. The mixture was precipitated and used for electroporation of Escherichia coli DH5.

The P1 phage 81-95, containing wild-type DNA from the 83B3-6 region on the cytological map (FlyBase http://fly base.bio.indiana.edu:82), was used to obtain an overlapping set of the fragments for cDNA library screens.

Two cDNA libraries were screened. One was prepared from eye imaginal disks of a wild-type stock and cloned into the EXlox vector (Novagen, Madison, WI). The second was prepared from 2-wk-old male and female wild-type adults (Canton S) in the ZAP II vector (Stratagene, La Jolla, CA). Both libraries were screened by standard protocols (SAMBROOK et al. 1989 ). Eventually, 80,000 phage from the eye imaginal disk cDNA library and 1,200,000 phage from the adult cDNA library have been screened.

For sequencing, DNA fragments were cloned into the pSP72 (Promega, Madison, WI) or Bluescript II SK (Stratagene) vector. To obtain nested clones for sequencing, a gamma-delta transposon-based system was used (STRATHMANN et al. 1991 ). Sequencing was performed on a Sequi-Gen GT nucleic sequencing cell (Bio-Rad, Richmond, CA) using the Sequenase (v.2.0) kit (Amersham, Arlington Heights, IL).

Sequence analysis and multiple alignments were performed using DNA STAR software (DNASTAR, Madison, WI). Homology searches were performed at the National Center for Biotechnology Information's BLAST WWW Server.

Isolation and analysis of RNA:
RNA was prepared according to the method of CHOMCZYNSKI and SACCHI 1987 . Flies were homogenized in a solution of 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. A 0.1-volume of 2 M sodium acetate (pH 4.0), an equal volume of phenol, and a 0.2-volume of chloroform were added, sequentially. After centrifugation for 5 min and precipitation with isopropanol, the RNA was dissolved in 100 µl of homogenization solution and precipitated with an equal volume of isopropanol. The pellet was collected by centrifugation, washed with 70% ethanol, and stored at -70°. For Northern analysis, RNA samples were prepared in running buffer (20 mM morpholino propane sulfonic acid, 5 mM sodium acetate, and 1 mM EDTA) with 2.2 M formaldehyde and 50% (v/v) deionized formamide, and were then heat denatured for 5 min at 65°. The RNA was fractionated in running buffer on 1% agarose gels containing 2.2 M formaldehyde. The RNA was transferred by capillary blotting in 20x SSPE from the gel to neutral nylon membranes (QIAGEN, Chatsworth, CA) and was then hybridized to radiolabeled antisense RNA probes prepared as described before (BIRCHLER et al. 1990 ). The prehybridization and hybridization solutions were 0.5 M NaCl, 10% dextran sulfate, 0.2 mg/ml heparin, 1% sarkosyl, 0.1 M sodium phosphate (pH 7.0), 0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, and 50% (v/v) formamide. The filters were washed three times in a solution containing 5 mM sodium phosphate, 0.2% SDS, and 1 mM EDTA for 1 hr at 75°.

Antisense RNA probes were synthesized with T3, T7, or SP6 RNA polymerase (Promega). Description of the probes used here can be found in BIRCHLER et al. 1990 . The probe for the Vinculin gene is a cDNA cloned into the EcoRI site of the Bluescript II KS vector (ALATORTSEV et al. 1997 ).

RACE:
To obtain 5' and 3' ends for Rga, the RACE protocol was performed using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA) according to the manufacturer's recommendations. As a template for cDNA synthesis, the poly(A)⁺ RNA was isolated from Canton S adult flies using an oligo(dT) cellulose (SAMBROOK et al. 1989 ). For PCR, the following primers were used: KK-1, AGCTGTTGGTTAGCT GTGGGT GTC; and M3, CCCACAGCTAACCAACAGCT. The RACE products were directly cloned into pGEM-T vector (Promega).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and characterization of the P-element insertion l(3)03834:
To isolate autosomal genes involved in trans-regulation of white gene expression, the white-apricot allele was used to screen a set of lethal P-lacZ element insertions (KARPEN and SPRADLING 1992 ) available from the Indiana University Drosophila Stock Center. The apricot allele produces a leaky, hypomorphic phenotype that allows detection of a wide range of modulation of white expression. The molecular basis of the lesion is a parallel insertion of the retrotransposon, copia, into the second intron of white (GEHRING and PARO 1980 ; BINGHAM and JUDD 1981 ; O'HARE et al. 1984 ). Among several isolated P element insertions exhibiting both elevated and decreased pigment intensity on the white-apricot background, l(3)03834 has been chosen for further study. This insertion maps to chromosome 3 at 83B4-5 and yields a darkened w ^a phenotype.

To determine the phase of lethality, l(3)03834/TM3 flies were crossed to the wild-type stock Canton S and the progeny were mated together. The number of embryos, larvae, pupae, and adults were scored. As many as 89% of the embryos hatched, compared with 94% for Canton S; however, only 72% of third instar larvae were observed. This number was the same for pupae and adults. Thus, the lethality of l(3)03834 occurs at the embryonic and early larval developmental stages.

To confirm that the darker eye color and lethality attributed to l(3)03834 are caused by the same insertion, the P element was mobilized by supplying transposase with the 2-3 chromosome (ROBERTSON et al. 1988 ). Genomic DNA isolated from 116 independent rosy^- stocks was subjected to Southern and PCR-based analyses. About 25% of the established stocks contain internal deletions within the P element, but no external deletions encompassing sequences outside of the P element were found. The other 75% appear to be precise excisions at this level of analysis. Sequencing of the region containing the insertion site (see below) from one of those, l(3)03834^rev, shows that this reversion is accompanied by loss of the element and one copy of the 8-bp duplication, thus restoring the wild-type sequence at the insertion site. The chromosome carrying l(3)03834^rev was shown to be viable as a homozygote and shown to give a reversion of the eye color phenotype.

To characterize further and determine the nature of the effect of l(3)03834 on the white gene, the interaction of l(3)03834 with different white alleles, such as point mutations, transposable element insertions, lesions in the structural and regulatory regions, as well as an Adh promoter-white fusion construct, was examined. These tests were performed by crossing females carrying different white alleles to l(3)03834/TM3, Sb males. The eye color of the l(3)03834/+ males was compared to that of their TM3/+ brothers at the same age. If l(3)03834 affects white transcription, one would expect that some alleles altering the 5' region of white would not respond. On the other hand, if l(3)03834 is involved in pigment deposition, then the white alleles would not alter the effect and all such flies would show a difference. The results of these tests are summarized in Table 1.

In brief, l(3)03834 elevates the expression of the white gene in a subset of point and insertional mutations. Some are insertions of the retroelements, copia (w ^a; BINGHAM and JUDD 1981 ), blood (w ^bl; BINGHAM and CHAPMAN 1986 ), and coral (w ^co; A. CSINK, unpublished results) in the second or fifth intron of the white gene. Another affected allele, w ^IR1, results from the insertion of the retroposon I element in the fifth intron (SANG et al. 1984 ; FAWCETT et al. 1986 ). The same effect was found for most of the point mutations, which would imply that l(3)03834 acts by changing the amount of transcripts from the wild-type white promoter. Indeed, no interaction has been found with the w ^sp2 allele, which is a deficiency in the 5' regulatory region (ZACHAR and BINGHAM 1982 ). Another set of regulatory lesions unaffected by l(3)03834 are reversions of the original white¹ mutation. This mutation is caused by the insertion of a Doc element in the 5' leader of the white transcript (DRIVER et al. 1989 ). Three tested reversions, w ^eosin, w ^apricot-like, and w ^honey were shown to be secondary insertions in the Doc retroposon (O'HARE et al. 1991 ). The former two do not exhibit dosage compensation between males and females. Another nondosage compensating allele, w ^ivory, is also not affected. Finally, no response was found for the Adh promoter-white structural gene construct (BIRCHLER et al. 1990 ). Thus, the allele specificity tests reveal the necessity of the proper white regulatory sequences for interaction with l(3)03834. Indeed when the zeste¹ mutation represses white expression, a response to l(3)03834 no longer occurs.

In addition to the above-mentioned white alleles, a rearrangement chromosome, In(1)w ^m4h, was also tested. This inversion contains the white gene juxtaposed to the centromeric heterochromatin of the X chromosome, which causes white inactivation in a mosaic manner (REUTER and WOLFF 1981 ), referred to as PEV (SPOFFORD 1976 ; REUTER and SPIERER 1992 ; HENIKOFF 1996 ). l(3)03834 was found to suppress the mottled phenotype of the white gene in In(1)w^m4h. This suppression is not as strong as in the case of previously described suppressors [Su(var)s], which as heterozygotes usually give practically wild-type eye color (REUTER et al. 1990 ), but it is rather comparable in its strength with three other white modifiers, Wow, Lip, and Mow, which are also suppressors of PEV (BIRCHLER et al. 1994 ; CSINK et al. 1994 ; BHADRA and BIRCHLER 1996 ). To quantitate the effect of l(3)03834 on PEV, pigment assays were performed on the segregating populations (Table 2). Spectrophotometric measurement demonstrates that l(3)03834 significantly elevates pigment level in In(1)w^m4h; l(3)03834/+ males relative to their In(1)w^m4h; TM3/+ brothers. To exclude the possibility that the observed effect is caused by the presence of a preexisting hypothetical mutation that suppresses PEV other than the P-element insertion, the same analysis was done for revertant flies. The data presented in Table 2 show that the amount of pigment in In(1)w ^m4h ; l(3)03834^rev/+ and In(1)w ^m4h ; TM3/+ is similar.

To test whether the suppressing effect of l(3)03834 on the In(1)w^m4h chromosome is caused by a general suppression of PEV rather than a specific interaction with the white gene, the effect of l(3)03834 on a variegating allele of yellow in the inversion In(1)y^3P was examined. For segregating classes, l(3)03834/TM3 males were crossed to In(1)y^3P females, and the F₁ males segregating for l(3)03834 and the TM3 balancer were scored. The number of wild-type and yellow triple row bristles along the anterior margin of the wing blades were counted in each class of flies (Table 3). l(3)03834 reduces the frequency of yellow variegation among the In(1)y^3P flies thereby suppressing the yellow bristle variegation nearly threefold (3.3%) above the comparable control values (10.9%). On the contrary, the revertant flies show no suppression of yellow variegation. Thus, l(3)03834, but not the revertant, suppresses variegation of both white and yellow in two different rearrangements, implying that l(3)03834 is a general modifer of PEV.

Effect of l(3)0384 on the transcripts of unrelated genes:
Because l(3)03834 was implicated in the interaction with the regulatory sequences of the white gene, its effect on white transcripts was studied. To determine how general this effect is, the steady-state mRNA level of two related genes, brown (bw ; DREESEN et al. 1988 ) and scarlet (st ; TEARLE et al. 1989 ), together with four unrelated genes, rudimentary (r ; SEGRAVES et al. 1984 ), Phosphogluconate dehydrogenase (Pgd; SCOTT and LUCCHESI 1991 ), Glycerol phosphate dehydrogenase (Gpdh; VON KALM et al. 1989 ), and Vinculin (Vinc ; ALATORTSEV et al. 1997 ) were examined. RNA isolated from two different populations of adults segregating for the l(3)03834 and balancer chromosomes was subjected to Northern analysis. As a control for normal expression, the same analysis was performed for the revertant flies l(3)03834^rev. Triplicate RNA transfers were hybridized with probes for the genes mentioned above. A probe for the rp49 gene (KONGSUWAN et al. 1985 ) was used as a gel-loading control because, in the case of the mutation, there are no differences found for the level of rp49 transcripts when compared to rRNA (data not shown). The results of the phosphorimagery scans are given in Table 4. Briefly, the level of bw, st, and r expression is not significantly affected by l(3)03834. The other tested genes are affected but the level of their expression is usually greater in males than in females. For example, the transcripts of white in males are increased, while in females they are decreased. The level of Pgd and Gpdh mRNAs were found to decrease in both males and females. However, the most dramatic effect was observed for the Vinc gene. Although it is not affected in males, the transcripts in females are strongly decreased, which results in a very low amount of product. We considered the possibility that the decreased steady-state level of Vinculin mRNA is caused by an effect on the tissue or cells in which Vinculin is normally expressed. However, examination of mutant larvae and adult flies did not reveal any developmental changes of primordia or adult structures, particularly ovaries, testis, imaginal discs, and brain. Vinculin was shown to play an important role in processes of cell motility and adhesion as one of the main counterparts of linkages between actin filaments and transmembrane receptors (KEMLER 1993 ). The normal viability observed in flies with an extremely low expression of the Vinc gene confirms the previous results of the nonessentiality of Vinculin in Drosophila (ALATORTSEV et al. 1997 ). Thus, l(3)03834 exerts an effect on the steady-state RNA level, and this effect is not restricted to the white gene. The observation that four of seven tested genes respond to l(3)03834 significantly implies that l(3)03834 could be involved in general regulation of gene expression.

To study the effect of the l(3)03834 mutation on the white mRNA level in larvae and pupae, crosses were performed as described in MATERIALS AND METHODS. Developmentally staged samples were collected from third instar larvae, early, mid-, and late pupae. Triplicate RNA transfers were hybridized with white and rp49 antisense probes, the latter being used as a gel-loading control. The level of rp49 does not vary relative to ribosomal RNA (data not shown). The results of phosphorimagery analysis are shown in Table 5. There is an elevation of white transcripts in larvae in l(3)03834/+ compared to +/+ individuals. In middle pupae, the effect returns to control levels. The phenotypic elevation of white expression could be caused by a short pulse of increase, similar to the larval effect, that occurs between our sampled stages—most likely at the late pupal/early adult transition preceding the adult stage, where a slight but significant increase was found (Table 6).

Cloning of the Rga gene and analysis of cDNA clones:
To identify the gene responsible for the mutant phenotype, the genomic sequence flanking the P-element insertion was cloned via the plasmid rescue method. Screening a random and oligo(dT)-primed adult cDNA library yielded seven different clones falling into three regions, with the P element being inserted into the region of the 0.6-kb cDNA, c9.1 (Figure 1). A fragment of c9.1 revealed a single 2.8-kb mRNA on a Northern blot of wild-type RNA (Figure 2A). This mRNA will be subsequently referred to as the Rga mRNA. To study its transcription in the case of the mutation, a Northern blot analysis of total RNA from a segregating population of l(3)03834 and that of l(3)03834^rev was performed. In the case of the mutation, the amount of the Rga mRNA is decreased to one-half relative to the respective rp49 control in both males and females, and an aberrant 4.5-kb transcript appears (Figure 2D, lanes 3–4 and 9–10). The aberrant mRNA could result from termination of transcription within the P element, particularly within the hsp-70 tail (MLODZIK and HIROMI 1992 ), which would give the transcript of the observed size. The transcript in the revertant l(3)03834^rev is restored to normal (Figure 2D, lanes 1 and 2 and 7 and 8). To study the transcription in mutant homozygotes, RNA was isolated from rare surviving flies, which do not carry the TM3 balancer marker Sb and, hence, are homozygous for l(3)03834, as well as from heterozygotes l(3)03834/TM3. The 2.8-kb Rga mRNA is not detectable in mutant homozygotes (Figure 2D, lanes 5–6 and 11–12), and the steady-state level of white mRNA is increased in mutant homozygous males (Figure 2D and Table 6). Sequence analysis confirmed that the insertion of a P element occurs within c9.1 (see below). Thus, the 2.8-kb mRNA appears to correspond to the gene responsible for the mutant phenotype because the insertion of a P element causes a twofold reduction of this mRNA in the heterozygotes and an absence in homozygotes while in the revertant the RNA returns to normal.

View larger version (13K): [in this window] [in a new window]   Figure 1. —Molecular map of the region containing the insertion site in l(3)03834. (A) Restriction map. Sites for endonucleases EcoRI (E), Nhe I (N), Pst I (P), Sal I (S), and XbaI (X) are shown. Site of the P[lacZ, rosy+] insertion in stock l(3)03834 is designated by a triangle. (B) Location, sizes, and intron–exon structure of the identified transcription units Rga and Atu. (C) Isolated cDNAs corresponding to Rga and Atu obtained by different methods: cDNAs isolated from cDNA libraries are shown by open boxes, and the RACE products for Rga mRNA are shown by lines with arrows on both sides. (D) Fragments of the Atu cDNA used for Northern analysis.

View larger version (28K): [in this window] [in a new window]   Figure 2. —Northern analysis of the region of P-element insertion in l(3)03834. (A–C) Hybridization of blot containing a wild-type RNA isolated from Canton S flies to antisense RNA probes synthesized from fragment of cDNA c9.1 (A), cDNA c9.3 (B), and fragment A of cDNA c9.16 (C). (D) Hybridization of a blot bearing total RNA from flies segregating for l(3)03834 in males (lanes 3–6) and females (lanes 9–12) and l(3)03834rev in males (lanes 1 and 2) and females (lanes 7 and 8) with antisense RNA probes for white, Rga, and Atu. Loading control (rp 49) is shown below.

The cDNA c9.1 is only 0.6 kb in length, implying that it is an incomplete copy. Because we failed to isolate a longer cDNA, a rapid amplification of cDNA ends (RACE; FROHMAN 1993 ) was performed. Using oligonucleotides M3 and KK1 (see MATERIALS AND METHODS) derived from the central region of clone c9.1, the 3' RACE product c9.17 and 5' RACE product c9.28, respectively, were obtained. The clone c9.17 was located to the region 0.7–6.8 kb on the restriction map, thus comprising the cDNA c9.14, while cDNA c9.28 was mapped to the 7.0–7.6-kb area (Figure 1). The size of the composite cDNA was found to be 2.8 kb, which is in good agreement with the size of the detected mRNA (see above). The cDNA c9.14, corresponding to the 3' end of the transcript, shows the same hybridization pattern as c9.1 when probed on a Northern blot (data not shown). Taken together, the data obtained from the cDNA and Northern analyses indicate that the cDNAs c9.14, c9.1, and the RACE products correspond to the same 2.8-kb mRNA.

Sequence analysis of the Rga transcript:
The cDNAs, RACE products, and appropriate genomic fragment were sequenced on both strands. A derived composite 2.8-kb cDNA was found to have an open reading frame of 1740 bp. When compared to the genomic sequence, Rga cDNA reveals the presence of seven introns, with the longest (1445 bp) separating the untranslated exon I from exon II. The insertion of the P element in l(3)03834 is located in exon I, and therefore before the translational start.

Conceptual translation of the open reading frame yields a predicted protein of 579 amino acid residues that has a molecular weight of ~59.4 kD (Figure 3A). The primary structure analysis reveals the presence of an opa or M repeat at position 61–71. This repeat is composed of (CAA)_n or (CAG)_n, and it encodes a stretch of polyglutamine as it does in Notch (WHARTON et al. 1985 ). Scattered polyglycine tracks were found within regions at positions 126–151 and 262–361.

View larger version (69K): [in this window] [in a new window]   Figure 3. —(A) Predicted protein sequence of RGA. (B) Multiple alignment of the predicted amino acid sequence of the C-terminal domain of RGA with that of the global-negative transcription factor Not2p from Saccharomyces cerevisiae (yeast); its homolog, gene B0286.4 (EMBL/GenBank accession number U39848), from C. elegans (nematode); and two translated human EST from Homo sapiens [EMBL/GenBank accession numbers D17177 (human 1) and H96804 (human 2)]. Amino acids that are identical between RGA and one or more of other proteins are shaded. The alignment was made by use of the computer program MegAlign from DNASTAR.

A sequence database search using the BLAST program (ALTSCHUL et al. 1990 ) against GenBank sequences translated in the conceptual six frames failed to reveal any significant similarity. All matches resulted from the presence of polyglutamine and polyglycine tracks in query. When the truncated RGA sequence without the 200 amino acid residues from the N-terminus containing these tracks was used in a similarity search, however, the homology to the yeast global negative transcription factor Cdc36p (Not2p; COLLART and STRUHL 1994 ) was found (Figure 3B). Even stronger homology was revealed to the Caenorhabditis elegans gene BO286.4 (EMBL/GenBank accession number U39848). Two other homologues are human expressed sequence tags (ESTs; EMBL/GenBank accession numbers D17177 and H96804). Potentially due to the fact that ESTs were sequenced on one strand only, there are frameshifts in comparison to the predicted sequence of RGA.

Analysis of the Atu transcript:
As mentioned above, in the course of cDNA library screens using the genomic fragments covering 14 kb around the insertion site, two classes of cDNAs were isolated, with one of them corresponding to the Rga gene (Figure 1). Another class of cDNA was found to map between positions 7.7 and 10.2 kb and defines Atu (Another transcription unit). Taking into account the close proximity of Atu and the P-element insertion site (Figure 1), the question was addressed whether the product of Atu could be responsible for the mutant phenotype of l(3)03834. To answer this question, a molecular characterization of Atu was also performed.

Deduced from DNA sequence analysis of overlapping cDNAs and genomic fragments, the Atu transcribed sequence consists of four exons and three small introns. Two nonoverlapping cDNA fragments were used for a Northern analysis. One of them is a cDNA c9.3, virtually corresponding to exon IV, while another one is fragment A, containing the sequences of exon III (Figure 1D). When the cDNA c9.3 (1.4 kb) was probed on a blot containing wild-type RNA, a minor (2.4 kb) and a major (1.4 kb) transcript were detected (Figure 2B). On the contrary, fragment A reveals only the 2.4-kb band and does not detect the 1.3-kb mRNA (Figure 2C). Thus, Atu produces two nested mRNAs overlapping in their respective 3' ends.

The 5' end of the 2.4-kb mRNA is located in close proximity to the P-element insertion site. To investigate whether it affects the transcription of the 2.4-kb mRNA, a Northern analysis in a segregating population of l(3)03834 was performed. As seen in Figure 2D and in Table 6, the transcription of the 2.4-kb Atu mRNA is not significantly affected in the case of the P-element insertion in both heterozygotes and homozygotes.

The derived composite Atu cDNA contains an open reading frame capable of encoding a protein of 724 amino acid residues (Figure 4). Several noteworthy features were found in the predicted amino acid sequence. First, a central portion of the protein between residues 395 and 595 reveals 27% identity to Leo1p from yeast (EMBL/GenBank accession number P38439; data not shown). The biological function of LEO1 is unknown; however, its product was shown to be nonessential for cell viability, as indicated by the absence of a phenotype from gene disruption mutations (MAGDOLEN et al. 1994 ). Second, the N-terminal 240 residues of ATU are 49% arginine plus serine. Many of these are present as arginine-serine or serine-arginine repeats. This feature is characteristic of an RS domain, which is found in a large group of related proteins that play an important role in spliceosome assembly and in 5' splice site selection (for review see HOROWITZ and KRAINER 1994 ; FU 1995 ; ADAMS et al. 1996 ). The RS domain appears to participate in protein-protein interaction and is involved in directing the protein to nuclear speckles, the specific subnuclear compartment where splicing occurs (LI and BINGHAM 1991 ; KOHTZ et al. 1994 ; for review, see MANLEY and TACKE 1996 ). It is unlikely, however, that the ATU protein is involved in splicing metabolism. Most proteins containing an RS domain also possess one or several RNA recognition motifs (BIRNEY et al. 1993 ); the ATU protein does not. Also, ATU does not have the amino acid motifs that are necessary and sufficient for directing a protein to nuclear speckles. This motif comprises a nucleoplasmin-like nuclear localization signal and a repeating arginine-serine dipeptide sequence adjacent to a short stretch of basic amino acids (HEDLEY et al. 1995 ).

View larger version (40K): [in this window] [in a new window]   Figure 4. —Predicted amino acid sequence derived from the composite cDNA of Atu. Nucleoplasmin-like nuclear localization signal (ROBBINS et al. 1991 ) is in italics. Multiple serine-arginine and arginine-serine repeats are underlined.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this report, a newly recognized Drosophila gene, Rga, is described. It modulates white steady-state mRNA level differentially depending upon the developmental stage. In addition, it affects the transcript levels of several other related and unrelated genes, sometimes in a strongly sex-specific manner. Rga also acts as a suppressor of PEV. The data presented here demonstrate that these phenotypes result from a loss of function mutation of a gene containing a domain homologous to the yeast general transcription regulator CDC36 (NOT2).

Initially, Rga was isolated in a large screen for autosomal recessive lethal P-element insertional mutations exhibiting an interaction with the white gene. A darker eye color was produced in the presence of an insertion on chromosome 3, l(3)03834, and precise excision of the P element eliminated the eye color effects, lethality, and the other consequences as well.

There are two transcription units, Rga and Atu, located head to head near the P element insertion site. Although the P element insertion is near Atu, it is actually inserted in the transcription unit of the adjacent gene, Rga, and the normal transcription of Atu is not significantly affected by l(3)03834. This finding disfavors the possibility that Atu is responsible for the mutant phenotype in l(3)03834.

The data presented here argue that l(3)03834 is a loss of function allele of the Rga gene. The insertion of the P element occurs within the 5' untranslated region of this mRNA, thus disrupting its transcription. The level of normal Rga mRNA is decreased by one-half in l(3)03834 heterozygotes and is not detected in mutant homozygotes, while the revertant has a normal level of these transcripts.

Several lines of evidence suggest that Rga encodes a protein involved in transcriptional regulation. The deduced amino acid sequence of RGA reveals a pronounced domain structure. The N-terminal domain is enriched in polyglutamine and is separated by polyglycine stretches from the NOT2-like, C-terminal domain. Polyglutamine repeats are considered to be common to transcription factors (BENEDYK et al. 1994 ). They are found in both transcriptional repressors and activators, and their primary function is to interact specifically with other components of the transcriptional apparatus (for review, see TJIAN and MANIATIS 1994 ; HANNA-ROSE and HANSEN 1996 ). The C-terminal domain of the RGA protein shows significant similarity to the general negative regulator of transcription in yeast, Cdc36p (Not2p) (COLLART and STRUHL 1994 ). The Not2p-like domain is highly conserved, being present not only in yeast and Drosophila, but also in humans and nematodes. The polyglycine stretches located in the central portion of the protein potentially could serve as a hinge between the two domains, allowing flexibility in their relative positions.

The features attributed to the Rga gene seem to be rather unusual. Its loss of function allele exerts both positive and negative effects on the expression of the white gene, depending upon the developmental stage. Thus, increases or decreases of gene expression could be governed by a single gene. These developmental differences have been reported previously for three other white modifiers, Inr-a (RABINOW et al. 1991 ), Wow (BIRCHLER et al. 1994 ), and Mow (BHADRA and BIRCHLER 1996 ). However, Rga not only affects white, but also causes modulation of expression of several other tested genes. The most striking example is its strong sex-specific effect on the X-linked Vinculin gene. Although the transcription in males is normal, in females, it is severely decreased in the presence of the Rga mutation.

Perhaps the most remarkable feature of Rga is its direct or indirect involvement in regulation of chromatin function, which follows from its suppression of PEV. Three genes previously identified in a search for modifiers of white expression, Wow (BIRCHLER et al. 1994 ), Mow (BHADRA and BIRCHLER 1996 ), and Low (BHADRA et al. 1997A ), were also recognized as weak modifiers of PEV. In addition, some genes isolated as modifiers of PEV are required for normal expression of homeotic genes. One of them, Trithorax-like, encodes the GAGA transcriptional factor that binds to the promoters of several genes and stimulates their transcription (FARKAS et al. 1994 ). Another example, E(var)3-93D, was also shown to display homeotic effects. It encodes a chromosomal protein with a domain homologous to several transcriptional regulators (DORN et al. 1993 ). It was proposed that the latter two genes are involved in establishing or maintaining an open chromatin configuration that is required for transcriptional initiation.

There are two general models that could explain how Rga differentially regulates the transcription of many unrelated genes and suppresses PEV. Rga could interact with the components of the general transcriptional machinery. This mechanism was proposed for CDC36 (NOT2) in yeast. CDC36 (NOT2) has been suggested to act as a negative regulator that directly affects the utilization of the TATA element (COLLART and STRUHL 1994 ). But, unlike CDC36 (NOT2), loss of function of Rga results in both negative and positive regulation of transcription of the target genes. This paradox could be resolved if Rga acts indirectly by affecting the expression of various other factors which, in turn, are involved in regulating gene activity both as repressors and activators. Among the loci regulated by Rga could be genes coding for components of heterochromatin, which could explain the suppression of PEV in the case of mutation in Rga.

In the second model, Rga acts on the establishment or maintenance of the appropriate chromatin state in the promoter region. Direct or indirect involvement of the RGA protein in regulation of chromatin structure follows from the finding that Rga acts as a weak suppressor of PEV. Since RGA does not possess any known DNA-binding motifs, however, it is unlikely to affect chromatin structure directly. Instead, it contains the glutamine-rich domain that could be responsible for protein-protein interactions (for review see TRIEZENBERG 1995 ). Also, the Not2p-like domain, as proposed previously (COLLART and STRUHL 1994 ), could inhibit the basic transcription factor(s). One can suppose that Rga mediates the interaction between the components of chromatin and the RNA polymerase II transcriptional complex and/or transcriptional factors. On the other hand, it was demonstrated that the Not2p protein together with three other proteins, Not1p, Not3p, and Not4p, form a multiprotein complex in yeast (COLLART and STRUHL 1994 ). Homology searches against EST databases revealed the presence of both Not1p and Not4p homologs in humans, and Not4p in mice and nematodes. It is therefore likely that the Not proteins exist in Drosophila, and that RGA could interact with them in a complex via the glutamine-rich domain, for example. In this case, a putative Not4p containing two zinc fingers (CADE and ERREDE 1994 ; IRIE et al. 1994 ) could mediate interaction between the RGA protein and the DNA of the promoter region of a target gene.

	ACKNOWLEDGMENTS

We thank the Indiana University Drosophila Stock Center for providing flies of l(3)03834. We are grateful to DMITRY NURMINSKY and MARIA NURMINSKAYA for help in cloning the RACE products, DAN HARTL for sending the P1 clone, and PAUL MAHONEY for providing a cDNA library. Special thanks to members of the Birchler lab for discussion and critical comments. This study was supported by a National Science Foundation grant to J.A.B. M.V.F. was supported by a postdoctoral fellowship from the Molecular Biology Program at the University of Missouri-Columbia. The sequence reported here was deposited in EMBL/GenBank (accession number U75467).

Manuscript received November 11, 1996; Accepted for publication October 1, 1997.

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