INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The development of multicellular organisms, even at the level of a single cell, demands a complex array of transcriptional regulation mechanisms for proper proliferation and development. To meet the demand, eukaryotic cells utilize transcriptional machinery comprising dozens of proteins that recognize and initiate RNA synthesis from promoters and that regulate the efficiency of transcription using thousands of specialized transcription factors. In addition, a number of coactivator complexes working at diverse stages of transcription add to the depth of regulatory complexity to achieve the orchestrated developmental control of gene expression in higher eukaryotes. Although these coactivator proteins appear to be required for transcriptional regulation in general, different groups of genes show different coactivator requirements. In addition, these coactivator functions are carried out by a number of complexes. Therefore, each coactivator complex appears to have unique and specific roles in transcriptional regulation of diverse developmental processes.

Two major coactivator complexes, TFIID (20, 38) and Mediator (24, 25), integrate and relay diverse regulatory signals to the basal transcription machinery through their association with TATA-box binding protein (TBP) and RNA polymerase II (pol II), respectively. Both complexes were shown to be required for transcriptional activation in an in vitro transcription system under specific conditions (2, 3, 17, 32). Depletion or inactivation of TFIID-specific TBP-associated factors affects the transcription of a subset of the genes involved in cell cycle regulation and development, suggesting that TFIID may act as a gene-specific rather than a general coactivator (21, 33, 47).

The Mediator complex was first identified in budding Saccharomyces cerevisiae as a general intermediary complex that mediates signal transfer between transcriptional activator proteins and the basal transcription machinery (24, 25). The search for similar Mediator complexes in mammalian systems led to the identification of a number of homologous complexes (5, 15, 18, 23, 35, 39, 41, 45). These complexes contain more than one Mediator homolog and share several components, but their overall compositions are different from each other and from that of the yeast Mediator complex.

Biochemical analysis of the yeast Mediator complex revealed that it is composed of several functional modules, each of which regulates distinct groups of genes (28, 30, 34). Mutations in the Gal11 and Med10 proteins caused severe transcriptional defects specifically to the genes involved in carbon metabolism (e.g., GAL1) and amino acid synthesis (e.g., HIS4), without affecting the expression of other groups of genes (19). The distinct activator-specific binding regions of Mediator underlie the gene-specific regulatory mechanism of Mediator subunits (37). In addition, alleles of gal11, sin4, and rgr1 affect the process of transcriptional repression as well as activation (11, 42). In vitro transcriptional analysis of mammalian Mediator homologs also demonstrated the requirement for the Mediator complexes for both positive and negative regulation of transcription (18, 45). Compared to the extensive genetic analysis of the Mediator complex in yeast, the functional analysis of Mediator genes in multicellular organisms is currently limited. Analysis of evolutionarily conserved subunits of Mediator (Med6, Srb7, Med7, and Med10) with the use of an RNA interference assay revealed that Caenorhabditis elegans Mediator homologs are required for transcriptional activation of developmentally regulated genes (26). These conserved subunits of Mediator complexes appear to have similar roles in mammals as well: murine Srb7 is essential for embryonic stem cell viability and development (46). On the other hand, disruption of a metazoan-specific subunit of Mediator revealed a gene-specific function. The C. elegnas Trap230 gene was shown to regulate lineage-specific expression of transcription factors (51). Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and impaired neuronal development (22). Clonal analyses of Drosophila melanogaster Trap80 and Trap240 mutants revealed their functions in the specification of adult cell and segment identity (4). Therefore, the metazoan Mediator subunits appear to contribute their gene- or activator-selective functions to diverse developmental processes.

To pinpoint the physiological functions of Mediator homologs in higher eukaryotes, we isolated mutants for a Drosophila homolog of yeast Med6 (dMed6) and examined their effects on development and transcriptional activation. Our results suggest that dMed6 is essential for cell viability and/or proliferation of diverse germ line and somatic cells and is required for transcriptional activation of a subset of genes involved in diverse aspects of development. Therefore, dMED6 appears to play an important role as a gene-specific transcriptional coactivator in Drosophila as does Med6 in yeast.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Degenerate PCR-based cloning of Drosophila Med6 homolog. To clone a Drosophila Med6 homolog, degenerate PCR primers were designed for the conserved regions of yeast, C. elegans, and human Med6 proteins and used to amplify Drosophila embryonic cDNA. Sequencing analysis of the fragments amplified with diverse sets of the Med6 degenerate PCR primers revealed that primers M1-1 (5'-cggaattcGTN TTR GAY TAY TTT-3') and M5-1 (5'-cgggatccDAT DAT RTA RTA RTC-3') amplified a fragment with a sequence homologous to those of other Med6 genes (lowercase letters indicate the restriction enzyme site incorproated at the end of each PCR primer). By using the PCR fragment as a probe, cDNA and genomic DNA clones were isolated from screens of a Drosophila adult ZAPII cDNA library (Stratagene) and a Drosophila genomic Charon 4A genomic DNA library, respectively. Three independent cDNA clones and the 4.2-kb BamHI-EcoRI genomic fragment identified by Southern analysis to contain the dMed6 gene were sequenced and analyzed for gene structure with the use of the GCG program (Wisconsin package). The sequence and structure of the dMed6 gene (CG9473) were described in the Genome Annotation Database for Drosophila (http://www.fruitfly.org).

Preparation of larval nuclear extracts. Whole animals (0.l ml), carrying dMed6^26/26 and dMed6^26/+, at the late-third-instar larval stage were resuspended in 0.25 ml of NEB(0.3) (0.3 M sucrose, 10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.1 mM EGTA, 0.5 mM dithiothreitol, 1× protease inhibitor), homogenized with a micropestle, and filtered through synthetic cotton. Filtered supernatant was loaded on 0.25 ml of NEB(1.7) [same as NEB(0.3) except 1.7 M sucrose] and centrifuged for 15 min at 12,500 rpm and 4°C. Nuclear pellets were resuspended in NEB(0.3) for immunoblotting or in 0.1 ml of HEMG-0.4K (25 mM HEPES-KOH [pH 7.6], 400 mM potassium acetate, 5 mM magnesium acetate, 0.1 mM EDTA, 5 mM -mercaptoethanol, 20% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride). Nuclei resuspended in HEMG-0.4K were disrupted by three cycles of freezing and thawing in liquid nitrogen and a water bath. After the lysates were centrifuged for 15 min at 15,000 × g and 4°C, the supernatants were collected and used for immunoprecipitation with anti-dSOH1 antibody beads or subjected to Superose-6 chromatography.

Transcriptional and biochemical analyses. Nuclear extract (Drosophila Oregon R embryo) preparation, in vitro transcription, immunodepletion, and gel filtration analyses were carried out as described previously (36). The antibodies against each Drosophila Mediator protein used in this study were described previously (36).

EMS mutagenesis. Two hundred 3-day-old isogenic w¹¹¹⁸ male flies were fed with ethane methyl sulfonate (EMS) solution (25 mM) as described by Huang and Baker (21a) and crossed with an equal number of TM3 Sb Ser virgin female flies to balance the third chromosome with TM3 Sb Ser. Each of 6,555 males carrying the EMS-mutagenized third chromosome over the balancer was crossed individually with three Df(3R)by10/TM3 Sb females. After 15 days, the lines without viable Sb⁺ progeny were selected, and the balanced males from each of the selected lines were crossed with Df(3R)B104/TM3 females to identify the lines that produce viable Sb⁺ progeny. Thirty-six lines suffering lethality whose genes were isolated within the nonoverlapping regions of the Df(3R)by10 and Df(3R)B104 chromosomes were crossed to each other to identify the complementation groups. A single male from each complementation group was crossed with w¹¹¹⁸; p{w⁺=dMed6 (10.4 kb)}/CyO; Df(3R)by10/TM3 females, and the viability of the w¹¹¹⁸; p{w⁺=dMed6 (10.4 kb)}/+; +*/Df(3R)by10 progeny was examined to identify the dMed6 mutants.

SSCP analysis. The transcribed region of dMed6 was amplified in three fragments of 300 to 400 bp from wild-type and mutant heterozygous genomic DNAs. Each PCR product was cloned in pBluescript SK(+) vector (Stratagene). Ten independent clones were amplified for each PCR fragment and displayed along with the corresponding wild-type PCR fragment on 8% nondenaturing polyacrylamide gels (acrylamide-bisacrylamide ratio, 49:1) with 10% glycerol for 12 h at room temperature or 4°C and visualized by silver staining. The PCR products that showed abnormal migration on the single-strand conformational polymorphism (SSCP) analysis were sequenced to find the mutations.

FLP-FRT-mediated clonal analysis. Clones of mutant cells were generated by the Flip recombinase-FRT site (FLP-FRT)-mediated mitotic recombination system (49). y w hsFLP/+; FRT-82B p{w^+mC=ovo^D1-18}3R1 p{w^+mC=ovo^D1-18}3R2/FRT-82B dMed6²⁶ and y w hsFLP/+; FRT-82B p{w^+mC=ovo^D1-18}3R1 p{w^+mC=ovo^D1-18}3R2/FRT-82B ry⁵⁰⁶ females (10) were generated by standard crosses. For germ line clonal analysis, 200 female flies (3 to 5 days old) were heat treated at 37°C for 2 h during the late-first-instar larval stage and mated with 50 w¹¹¹⁸ male flies. After 2 days embryos were collected at 4-h intervals. For twin spot analysis of adult tissues, dMed6 clones were monitored using the associated marker w in eyes and the green fluorescent protein (GFP) marker expressed from the ubiquitin-63E (Ubi) promoter in imaginal discs and ovarian follicle cells. For these experiments, y w hsFLP/+; FRT-82B P{w^+mC=Ubi-GFP}/FRT-82B dMed6²⁶ and y w hsFLP/+; FRT-82B P{w^+mC=Ubi-GFP}/FRT-82B ry⁵⁰⁶ females were generated. Clones were induced by heat shock (2 h, 37°C) during the first or second instar. Imaginal discs were dissected from late L3 larvae. Eyes and ovaries from 2- to 5-day-old adult females that were heat shocked for 2 h at the end of the late-first-instar larval stage were analyzed. The dissected imaginal discs and ovaries were fixed with 4% formaldehyde for 20 min at 22°C. GFP expression was analyzed by confocal laser microscopy (Bio-Rad; MR1024).

GFP in larval tissues. Larval tissues were dissected and mounted in 1× phosphate-buffered saline (PBS). Whole larvae were etherized in 20% (vol/vol) diethyl ether in ethanol for 5 min, mounted in a 70% (vol/vol) glycerol (in PBS) on a standard slide glass with a paper tape support for a standard coverslip, and viewed directly. Samples were observed with a plane fluorescence microscope (Carl Zeiss; Axioscop 2) or confocal laser scanning microscope (Carl Zeiss; LSM510) under Hg illumination with standard fluorescein isothiocyanate fluorescence filters for the observation of modified GFP^S65T.

DNA chip analysis. cDNA expression profiles of 192 genes were analyzed by microarray analysis on a polylysine-coated glass slide as described previously (48) with the following modification. Total RNA was isolated from whole flies carrying dMed6^26/26 and dMed6^26/+ at the late-third-instar larval stage. Fluorescent cDNA was produced with 5 µg of total RNA, oligo(dT) primers (Gibco-BRL), and Superscript II reverse transcriptase (Gibco-BRL) in the presence of Cy3 or Cy5 fluorescence-tagged dUTP (Amersham-Pharmacia). The labeled cDNA was dissolved in 3× saline sodium citrate buffer (SSC; 1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and hybridized to microarrays for 12 to 16 h at 65°C in humidified incubation chambers. Arrays were then washed for 5 min in 0.6× SSC-0.03% sodium dodecyl sulfate and rinsed for 10 min in 0.06× SSC, spun dry, and scanned with a confocal laser array scanner (GSI Lumonics; Scanarray Lite). The results were analyzed with Quantarray (GSI Lumonics) and Gene Spring, version 3.1 (Silicon Genetics). Results from two independent hybridization analyses were averaged.

Quantitative reverse transcription-PCR (RT-PCR). Total RNAs (5 µg) isolated from dMed6^26/26 or dMed6^26/+ flies at the late-third-instar larval stage were incubated with oligo(dT) primers (Gibco-BRL), Superscript II reverse transcriptase (Gibco-BRL), and deoxynucleoside triphosphate (dNTP; Boehringer Mannheim) in 30-µl reaction buffer supplied by the manufacturer. The specific gene of interest was amplified with 1 µl of the cDNA synthesized in the presence of a mixture containing 5 pmol of specific primers, 1 µl of a 10× SYBR Green I solution (Roche), 2 µl of 10× PCR buffer (100 mM Tris-Cl [pH 8.8], 500 mM KCl, 25 mM MgCl₂, 1% Triton X-100), 1.6 µl of 2.5 mM dNTP mixture, and 15 U of Taq DNA polymerase in a 20-µl reaction mixture. The product was amplified by 35 cycles of PCR (30 s at 94°C, 30 s at 63°C, and 1 min at 72°C). The incorporation of the dye into the amplified products was monitored by iCycler (Bio-Rad), and the concentration of a specific transcript in the sample was analyzed by the associated software based on the standard curves predetermined with known amounts of target transcripts. Quantities of rp49 gene transcripts were used as a total-cDNA input control. Results from three independent RT-PCR analyses were averaged.

LacZ activity staining in larval tissues. Enhancer trap LacZ lines (43) (provided by the Bloomington Stock Center) were crossed with a dMed6²⁶ mutant to make p{ry^+t7.2=PZ}*/+; dMed6²⁶/TM6B Tb p{w^+mC=Ubi-GFP} flies, and their males were crossed with dMed6²⁶/TM6B Tb p{w^+mC=Ubi-GFP}virgin females. Larval progeny were washed with water extensively and stored in 1× PBS. Tissues were dissected in cold 1× EBR (130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM HEPES-NaOH [pH 6.9]) and then fixed within 5 min with fixative (4:1:1 ratio of water-37% formaldehyde-buffer B [100 mM KH₂PO₄-K₂HPO₄ {pH 6.8}, 450 mM KCl, 150 mM NaCl, 20 mM MgCl₂)]). The fixed tissues were stained with LacZ staining solution {10 mM NaH₂PO₄-Na₂HPO₄ [pH 7.2], 150 mM NaCl, 1 mM MgCl₂, 3.1 mM K₄[Fe²⁺(CN)₆], 3.1 mM K₃[Fe³⁺(CN)₆], 0.3% Triton X-100, 0.05% X-Gal [5-bromo-4-chloro-3-indolyl--D-galactopyranoside]} at 37°C for 16 h. The stained tissues were dissected on a slide and mounted in 1× PBS, and the coverslip was sealed with nail polish. The samples were analyzed by using Nomarski images (Carl Zeiss; Axioscope 2).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Cloning and expression of Drosophila Med6 To study the function of Mediator in Drosophila development, we cloned a Drosophila Med6 homolog by PCR. We used degenerate primers based on the conserved regions of the yeast, nematode, and human MED6 proteins (Table 1). The Drosophila Med6 homolog was isolated and termed dMed6, and it encodes a 247-amino-acid polypeptide (GadFly CG9473). This predicted polypeptide has 43 and 19% identity to human and yeast MED6, respectively. Sequence analysis of the dMed6 cDNA and genomic clones revealed that the dMed6 gene is composed of four exons and controlled by a distal promoter element-containing TATA-less promoter. In situ hybridization of the dMed6 cDNA to polytene chromosomes revealed that dMed6 is located on the third chromosome at the 85E10-13 locus, where the dMed6 sequence was identified in the Drosophila genome project.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   Expression of dMed6. (A) Developmental Northern analysis of dMed6. Poly(A+) RNA (3 µg) isolated from each indicated developmental stage was analyzed with a dMed6 cDNA probe. The amount of rp49 transcript is shown as a loading control. E, embryo; L, larva; PP, prepupa; P, pupa; A, adult. (B) Immunostaining of ovaries and embryos with anti-dMED6 Ab (red) or Syto16 (green; nucleic acid).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Association of dMED6 with Mediator complex. (A) Coimmunoprecipitation of dMED6 with other Mediator subunits. Drosophila embryo nuclear extract was immunoprecipitated with anti-dSOH1 Ab, and the input (I), supernatant (S), and pellet (P) were analyzed by immunoblotting with Abs against the proteins indicated at the left. (B) Gel filtration analysis of dMED6-containing complex. Embryo nuclear extract was put on a Superose-6 gel filtration column, and the filtrates were analyzed with Abs against the proteins indicated at the left. The input and the elution positions of size markers are marked. (C) Transcriptional activation of the E4 promoter constructs by Gal4-VP16 in nuclear extracts. Before the in vitro transcription assay, the nuclear extracts were immunodepleted with anti-dSOH1 (-dSOH1) or anti--galactosidase (mock). Recombinant Gal4-VP16 (40 ng) was added to the reaction mixtures as indicated. Arrows, transcripts from the E4 templates containing five tandem Gal4 DNA binding sites (G5-E4).

Isolation of dMed6 mutants. Although the requirement of the Mediator complex for transcriptional activation has been well documented in yeast, there is little information about the physiological function of Mediator in higher eukaryotes. This prompted us to examine the gene-specific requirement of dMed6 for transcriptional activation and the role of dMed6 in developmental processes in Drosophila. To address these questions, we isolated dMed6 mutant alleles using mutagenesis induced by EMS (Sigma; M-0880).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Identification of dMed6 mutations. (A) SSCP analysis of dMed6 mutant alleles. PCR fragments (positions 61159 to 60748) amplified from wild-type (+), dMed626, and dMed664 mutant chromosomes were analyzed by SSCP gel electrophoresis. The extra band (arrowhead) and faster-migrating bands (arrows) are marked. (B) Mutation sites of dMed626 and dMed664. The structure of the dMed6 gene is marked with the mutations in each dMed6 mutant allele identified. dMed626 has one nucleotide change (G60947A) at the splicing acceptor of the second intron, and dMed664 has a five-nucleotide deletion (CACCG61049-5). Both mutations cause truncation of the dMED6 protein, as shown beneath. Gray boxes, wild-type coding regions; hatched boxes, nonauthentic amino acids added to the C-terminal ends of the mutant proteins due to the mutations. (C) Lethality of dMed6 homozygous mutants. The numbers of larvae, pupae, and flies viable after hatching from eggs were determined every day for wild-type (200 hatched larvae) and dMed626 mutant flies (168 hatched larvae) at 25°C. Most of the wild-type flies developed to adults in 10 days after hatching. However, a significant number of mutant flies died 3 to 5 days after hatching. The mutant flies showed no apparent developmental defect until the third larval instar but never developed to the prepupa stage.

Requirement of dMed6 for cell viability. To identify the developmental defects associated with the dMed6 mutations, we first determined the lethal phase of the dMed6 homozygous mutants. Among the embryos from dMed6²⁶/TM6B(GFP) and dMed6⁶⁴/TM6B(GFP) heterozygous flies, we collected dMed6 homozygous mutant embryos that do not express green fluorescence in the central nervous system (CNS). Developmental progress of these embryos was scored at 24-h intervals. Both types of dMed6 mutant embryos developed normally and reached third-instar larvae 2 days after hatching, as did the wild-type embryos. After two more days, all of the wild-type larvae quadrupled their size and had entered pupation, while the mutant larvae showed a slower growth rate and most of them died without pupating. A small number of mutant larvae survived for several more days but never developed into pupae (Fig. 3C). When we examined the dMed6²⁶/dMed6⁶⁴, dMed6²⁶/Df(3R)by10, and dMed6⁶⁴/Df(3R)by10 flies, we found that they all showed the third-instar larval lethality (data not shown). Therefore, dMed6 mutants are defective in a developmental process required for the transition from third-instar larva to pupa.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   dMED6-deficient Mediator complex. (A) Levels of dMED6 and dSOH1 proteins in wild-type and dMed626 mutant flies. Whole-cell extracts (20 µg) were prepared from wild-type and dMed626 embryos (E), first-instar larvae (L1), second-instar larvae (L2), early-third-instar larvae (L3-1), and late-third-instar larvae (L3-2). Proteins were immunoblotted with the antibodies indicated at the left. The actin protein was the loading control. (B) Immunoprecipitation of nuclear extracts of dMed6+/26 (W) and dMed626/26 (M) third-instar larvae with the anti-dSOH1 Ab. Equivalent amounts of the nuclear extract input (NE) and the immunoprecipitation pellet (IP) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the Drosophila Mediator Abs against the proteins indicated at the left (36).

Clonal analysis of dMed6 mutant. To confirm the requirement of dMed6 function for cell viability, we made dMed6 homozygous mutant cells with the FLP-FRT system (49). First, we used the FRT-dominant female sterile system (10) to examine the fate of dMed6 null germ line cells during oogenesis. Because ovo^D is a dominant female sterile mutation, the parental strain containing one copy of ovo^D is completely sterile. When wild-type homozygous clones without ovo^D were generated by the FLP-FRT system upon heat shock, the flies generated several fertile germ cells. However, when the dMed6²⁶ mutation was placed at a position trans-heterozygous to the site of ovo^D, mitotic recombination at a site proximal to dMed6 generated recombinant clones that were homozygous for dMed6²⁶ and that lacked ovo^D. The flies remained sterile even after the induction of mitotic recombination, which indicates that the dMed6²⁶ mutant clones were not able to generate mature germ line cells (Fig. 5A). Thus, dMed6 is needed for proliferation and/or development of germ line cells.

View larger version (79K): [in this window] [in a new window]   FIG. 5.   Clonal analysis of dMed626 homozygous cells. (A) Numbers of egg laid in 4 h by the female flies of the indicated genotypes with (+) and without () the induction of mitotic recombination in germ line cells by heat shock. The two hundred female flies (3 to 5 days old) of the indicated genotypes were heat treated at 37°C for 2 h during the late-first-instar larval stage and mated with 50 w1118 male flies for 2 days before egg collection. (B) Clonal analysis of dMed626 homozygous clones in somatic cells. The genotypes of the third chromosome, where the mitotic recombination was induced, are shown at the top. The boundaries of the twin spots are marked with solid (dMed6+) and dashed (dMed626) lines. Arrows and arrowheads, dMed6+ and dMed626 homozygous clones (two copies of w+ or GFP-expressing patches in eye, imaginal disc, and follicle cells), respectively. Clones were induced by heat shock (2 h, 37°C) during the first or second instar, and discs were dissected from late L3 larvae, whereas ovaries and eyes in 2- to 5-day-old adult females were examined.

dMed6 mutant phenotype. Although the growth rate of the dMed6 mutant decreased in the third instar, the mutant larvae continued to grow until they reached the size of fully grown wild-type larvae. Therefore, the death of dMed6 mutant larvae appeared to result from stage-specific developmental defects rather than simple growth defects. Dissection of the fully grown mutant third-instar larvae showed that most of the larval organs were a little bit smaller than wild-type organs (80 to 90% of wild-type size) but showed no other obvious abnormality. However, we were not able to detect antenna, wing, and leg imaginal discs in the mutant comparable in size to those in wild-type animals. The lack of obvious imaginal discs was intriguing and hinted at a specific requirement of dMed6 in imaginal disc development.

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Arrest of imaginal disc development in dMed626. (A) Effect of dMed6 mutation on the expression of Dll promoters. Whole mounts of wild-type and dMed6 mutant larvae containing a GFP expression construct under the control of the Dll promoter are shown for the indicated larval stages. GFP expression at the antennomaxillary complex is visible in the first-instar larvae of both the wild-type and dMed6 mutant (arrowhead). Organs with strong GFP expression are marked. ad, antenna imaginal disc; am, antennomaxillary complex; dd, dorsal T1 disc; sg, salivary gland; t1 to -3, leg imaginal discs; e1 to -3, thoracic ectoderm. (B) Dll induced GFP expression in CNS and imaginal discs. Dll expression patterns in the CNS and imaginal discs dissected from wild-type and dMed6 mutant third-instar larvae are shown. Arrowhead and arrows, eye-antennal imaginal discs (ead) and labial imaginal discs (ld), respectively. The GFP expression in thoracic neuromere cells (n1-3) is marked. ol, optic lobe. (C) Wild-type and dMed6 mutant wing imaginal discs dissected from second- and third-instar larvae. The wing imaginal discs dissected from third-instar larva were stained with LacZ driven by the dpp promoter. The second-instar wing imaginal discs are shown at higher magnification than the third-instar wing imaginal discs.

Expression profile analysis of dMed6 mutants. Studies with a mutant allele of yeast MED6 conferring temperature sensitivity revealed that MED6 is required for transcriptional activation of most but not all of the genes transcribed by pol II (21). To identify the genes responsible for the dMed6 mutant phenotype, the level of mRNAs in the dMed6 mutant and wild-type third-instar larvae was analyzed with microarrays. The microarray contained 192 different cDNA probes, which covered genes involved in diverse aspects of Drosophila development (Table 2). Wild-type and dMed6 mutant cDNAs labeled with Cy5 and Cy3 dyes, respectively, were hybridized simultaneously to the microarrays. The average of results from two experiment showed that 12% of the genes (22 out of 184 transcripts assayed) were down-regulated more than threefold, 27% (50 out of 184) were down-regulated two- to threefold, and the remaining 61% were changed less than twofold in the mutant compared with wild-type genes (Fig. 7A and Table 2). Genes required for metabolism (Glutamine synthetase 2 [Gs2], Larval serum protein 1 [Lsp1], Alcohol dehydrogenase [Adh], and cytochrome P450 [Cyp]), cell cycle control (cdc2 and Cyclin E [CycE]), and differentiation (Son-of-sevenless [Sos], oo18 RNA-binding protein [orb], and rolled [rl]) were down-regulated more than threefold in the mutant. In particular, the expression of developmentally regulated transcription factors (dorsal [dl], Hairless [H], apterous [ap], extra sexcombs [esc], dTrap240, and CG8609) and genes induced by the larval hormone (Hormone-receptor-like in 78 [Hr78], Ubiquitin-63E [Ubi-p63E], Heat shock protein 27 [Hsp27], and Ecdysone-inducible gene E2 [ImpE2]) was significantly reduced in the dMed6 mutant.

View larger version (32K): [in this window] [in a new window]   FIG. 7.   Expression profile of a dMed6 mutant. (A) Scatter plot analysis of microarray experiment. The hybridization intensities of the wild-type (x axis) and dMed6 mutant cDNA probes (y axis) are plotted on a log scale. rp49, trx, and genes down-regulated more than three-fold in the dMed6 mutant larvae are indicated. Diagonal lines, range of a twofold difference. Results from two independent hybridization experiments were averaged. (B) Comparison of expression levels of individual genes assayed by cDNA microarray (open bar) and quantitative RT-PCR (solid bar). Percentages of transcript in dMed6 mutant are compared to those for the wild type. Results from three independent quantitative RT-PCR experiments were averaged, and the deviations are marked with error bars.

Lsp1, cdc2, Adh, ap, brm, 18 wheeler

Requirement of dMed6 for tissue-specific transcriptional activation in vivo. Down-regulation of some genes involved in cell proliferation in the dMed6 mutant partly explains the mutant phenotype and suggested that other mitotically active larval tissues (gonad and neuroblasts) might also be affected by the dMed6 mutation. 4'-6-Diamidino-2-phenylindole (DAPI) staining of the larval ovary showed that the mutant ovary was significantly smaller and contained fewer cells than the wild-type ovary (Fig. 8A).

View larger version (68K): [in this window] [in a new window]   FIG. 8.   Requirement of dMed6 for the activation of developmental promoters. (A) DAPI staining of larval ovary. Arrowheads, ovaries. (B) Requirement for dMed6 for transcriptional activation in larval CNS. The expression of lacZ was driven by the promoters of the genes indicated at the left. (C) Effect of dMed6 mutation on the promoters of the genes listed at the left in the larval tissues indicated at the right.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The Mediator complex is generally required for most gene expression and functions in the recruitment of transcriptional machinery to promoters by activator-specific interaction of Mediator subunits (19, 21, 29). Therefore, the Mediator complex has an essential role in most developmental processes as a whole but still shows gene specificity in the requirement for each Mediator subunit. The distinct dMed6 mutant phenotype in most Drosophila cells reflects both the fundamental and the specific aspects of the complex in developmental regulation.

Although yeast Med6 plays a central role in the Mediator complex, only a distinct group of genes transcribed by pol II (15% of the yeast genome) requires Med6 activity (21). Consistent with this result, the microarray analysis of dMed6 mutants for transcriptional defects revealed that dMed6 is required for transcriptional regulation of a subset of genes in Drosophila as well. In particular, defects in cell proliferation and metabolism were most easily detected due to the down-regulation of several key regulators by dMed6 mutation. It is intriguing that Drosophila cdc2 and Cyclin-dependent kinase 7 (Cdk7), the essential regulators of cell proliferation, cause similar mutant phenotypes; larvae with mutations in these genes were also restricted in the mitotic proliferation of imaginal cells, while nonimaginal larval cells continued to grow and replicate their DNA (27, 44). In addition, the reduced level of CycE and brahma (brm) transcription may be partly responsible for the proliferation defects of the dMed6 mutant (14, 31). In addition to these, the transcriptional activation of Gs2 (16), Cyp (9), Adh (12), and Ecdysone-inducible gene L3 (ImpL3) (1), which are involved in the biogenesis of cellular components or removal of toxic metabolites, was severely reduced in the dMed6 mutants. Therefore, most of the genes expressed at high levels during the developmental transition from larva to pupa appear to require the function of dMed6 directly or indirectly for transcriptional activation.

Because the expression of these genes is highly stimulated by 20-hydroxyecdysone, dMed6 activity appears to be required for the mediation of regulatory signals from ligand-bound nuclear receptors to basal transcription machinery. In mammals, ligand-bound nuclear receptors (e.g., the vitamin D receptor and thyroid receptor) bind tightly to a mammalian Mediator homolog, the vitamin D receptor-interacting protein (DRIP)-thyroid receptor-associated protein (TRAP) complex (39, 50). The DRIP-TRAP complex has been suggested to activate transcription by recruitment of the Mediator complex to the promoter along with other transcription factors. Homologs for dMED6 and its associated Drosophila Mediator subunits (dSOH1 and dSRB7) are components of the DRIP-TRAP complex, suggesting that the mammalian MED6 homolog may function in transcriptional activation by nuclear receptors. However, the nuclear receptors interact with the Mediator complex via different subunits (e.g., DRIP205-TRAP220) (6, 40, 50). Therefore, dMED6 may function at the post-activator (nuclear receptor) binding stage in the relay of activation signals from ecdysone-induced nuclear receptors to basal transcription machinery, as does yeast Med6 in transcriptional activation by Gal4, which binds to the Gal11 subunit of the Mediator complex (30).

Although the microarray and quantitative RT-PCR analyses of whole mutant flies identified genes whose expression was affected by the dMed6 mutation, the examination of the tissue-specific expression of developmentally regulated promoters with LacZ and GFP reporters revealed several interesting details. First, dMed6 is required for activation of most but not all of the developmentally regulated promoters. Neither dpp expression at the optic lobe of the third-instar larval brain and imaginal discs nor esophagus-specific expression of zipper was defective, whereas a number of developmentally regulated promoters were inactive in the dMed6 mutants. Second, the transcriptional activation of a specific promoter was sometimes affected differently depending on where the gene was expressed. For example, the level of the Pka-C1 transcript measured by the microarray and quantitative RT-PCR decreased about 2-fold in the mutant but the Pka-C1::lacZ expression analysis for various tissues revealed transcriptional defects from 2- to 3-fold in muscles to more than 20- to 30-fold in salivary glands and brain. Transcriptional activation of the zip promoter was defective in the mutant salivary glands and was without abnormality in the esophagus. Similarly, transcriptional activation of the Dll promoter was completely lost in mutant imaginal discs, whereas a comparable level of Dll promoter activity was detected in the mutant CNS. Therefore, we conclude that dMed6 is required for gene-specific transcriptional activation of a group of genes required for diverse aspects of cell metabolism. However, whether all of these genes require dMED6 directly for transcriptional activation remains to be addressed.