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Department of Developmental Genetics, National Institute of Genetics, and Department of Genetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan
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Introduction |
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Abdominal-B (Abd-B) is a homeotic gene categorized to the bithorax complex. Abd-B specifies abdominal segments A5–A9 (parasegment (PS) 10–14). Abd-B encodes two proteins, Abd-Bm and Abd-Br, which share the C-terminal amino acid sequence including the homeodomain. Expression of Abd-B m products is regulated by iab5, iab6, iab7 and iab8 regions, which are implicated in the formation of A5, A6, A7 or A8 segments, respectively. Abd-B r products are expressed in the A9 segment and are required for genitalia development (Maeda & Karch 2006). In the present study, "Abd-B" represents Abd-B m class, transcribed from the Abd-B-RB promoter (Fig. 3).
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Previous studies have identified a unique supercoiling activity consisting of DNA supercoiling factor (SCF) and topoisomerase II isolated from the silkworm Bombyx mori (Ohta & Hirose 1990; Ohta et al. 1995). Subsequent study on Drosophila melanogaster SCF has revealed interaction with topoisomerase II in the nucleus and localization to puffs on polytene chromosomes (Kobayashi et al. 1998), suggesting a role for SCF in transcription on chromatin. The C-terminal region of SCF is essential for complex formation with topoisomerase II and supercoiling activity (Kobayashi & Hirose 1999). SCF is required for dosage compensation through male-specific hyper-transcription of X-linked genes (Furuhashi et al. 2006). However, SCF is localized to many sites on autosomes other than the X chromosome. The present study focused on the function of SCF on autosomes and demonstrated that SCF contributes to proper expression of Abd-B. Our findings illustrate a fundamental role for SCF in gene expression.
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Results |
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To obtain clues for clarifying the function of scf on autosomes, we introduced heritable scf RNAi (Furuhashi et al. 2006) and searched for a morphological defect. Most males died upon RNAi knockdown of scf, but a few surviving males reproducibly showed ectopic bristles on the ventral side of A6 (Fig. 1B). This is a sign of anterior transformation from A6 to A5, characteristic of functional deficiency of the Abd-B gene (Celniker et al. 1990). Indeed, we observed a similar phenotype in a heterozygote of Abd-BHCJ199(ry-,FRT-) harboring a P-element insertion in the promoter (Fig. 1C; Bender & Hudson 2000).
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To examine functional relationship between scf and Abd-B, we analyzed the genetic interactions. When we used the trangenic line UAS-IRscf[2–1] for RNAi, females were not lethal (Table 1, No. 1; see also Furuhashi et al. 2006). However, we found that in the same scf RNAi induced in the Abd-BHCJ199(ry-,FRT-) heterozygous background, viability of females (RNAi/Control) decreased to one-tenth that of wild-type females (Table 1, No. 2). Viability of females was not different between wild-type and the Abd-BHCJ199(ry-,FRT-) heterozygote (data not shown). These results suggest that scf functions in the same pathway as Abd-B. In addition, we noted that the observed interaction would be independent of the dosage compensation mechanism, as Drosophila dosage compensation does not occur in females.
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RNAi has limitations for the observation of adult males, since most males subjected to scf knockdown die during development. To overcome this situation, we isolated a loss-of-function mutant of scf. In the screening of Rac1 mutants, Ng et al. (2002) collected mutations that were lethal over Df(3)Rac1 lacking Rac1, Gef5 and scf, and carrying a deletion in the Rac2 gene (Rac2
), after EMS mutagenesis of a strain carrying Rac2. They then isolated lines that were rescued by expression of Rac1 from a transgene. One of the line (J2.4) that was not rescued by Rac1 expression was kindly provided by Drs Ng and Luo. We found that J2.4 includes a non-sense mutation in scf (scf1) (Fig. 2A). Recovery of scf1 was achieved through recombination between J2.4 and wild-type, followed by PCR screening for the absence of Rac2. In both sexes, scf1 is lethal at the third instar larval stage. This lethality was rescued by expression of genomic scf (Gscf) from a transgene. Heterozygotes of scf1 grew to adults, and did not show significant anterior transformations.
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Using the scf1 mutant allele, we investigated genetic interactions between scf and trans-regulators of Abd-B. We expected that scf would counteract Pc-dependent silencing of Abd-B, because scf RNAi causes anterior transformation in the posterior abdomen. In Pc3 heterozygotes, the male A4 tergite shows pigmentation as in A5 (Fig. 1E) (Duncan 1982). This phenotype represents posterior transformation caused by de-repression of Abd-B in A4 of the Pc mutant (Karch et al. 1994; Kopp et al. 2000; Jeong et al. 2006). We found that in the scf1 background, penetrance of A4 pigmentation due to Pc3 significantly decreased from 60% to 30% (Fig. 1F; Table 2A). Furthermore, expression of Gscf from a transgene significantly restored transformation frequency of double heterozygotes. The expression level of Pc did not increase in scf1 mutant (Supplementary Fig. S1). These data suggest that scf counteracts Pc-dependent silencing of Abd-B.
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Although scf1 heterozygotes did not show significant homeotic transformation, heterozygotes of trxG mutant alleles trxB11 and RpII140z43 cause anterior transformation in male A6 (Mortin et al. 1992; Milne et al. 1999). We examined the effect of combining the scf1 and trxG mutations on transformation phenotype. We observed a significant enhancement in penetrance of anterior transformation in double heterozygotes of scf1 and either trxB11 or RpII140z43 (Table 2B,C) compared with single heterozygotes of each. Furthermore, enhancement of transformation in double heterozygotes was significantly suppressed by introducing Gscf. Expression levels of trx and RpII140 did not decrease in scf1 mutant (Supplementary Fig. S1). These results indicate that scf works together with trxG for the maintenance of Abd-B expression and is involved in the segment-specific patterning of the abdomen.
Reduced expression of Abd-B in scf 1
To examine the influence of scf1 on expression of Abd-B, we measured expression levels of Abd-B using quantitative RT-PCR. We prepared embryos that were maternally and zygotically homozygous for scf1 utilizing the germline clone (GLC) technique (Chou et al. 1993). We compared scf1 GLC homozygotes with scf wild-type embryos at 5–16 h after egg laying (AEL). Levels of Abd-B gene products were reduced by ~30% in scf1 homozygotes as compared to wild-type. By contrast, expression of control genes β1-tubulin and Rp49 were unaffected by the scf1 mutation. We also observed decreased expression of Abd-B in prepupae of the transgenic line UAS-IRscf[2–1] upon Actin 5C promoter-driven Gal4 expression (data not shown). These data indicate that SCF positively regulates Abd-B expression.
Occupancy of SCF in Abd-B promoter
If SCF directly regulates Abd-B expression, it should be present in the regulatory region of Abd-B. To analyze SCF localization around the Abd-B gene loci, we performed chromatin immunoprecipitation (ChIP)-on-chip experiments. Solubilized chromatin fragments were prepared from yw embryos, followed by immunoprecipitation with anti-SCF antibodies to create hybridization probes. These probes provided fine results in hybridization. Raw data were processed using Feature Extraction software (Agilent Technologies, Palo Alto, CA). We found that significant SCF signals were present in the Abd-B promoter region (Fig. 3). Signals located around 1.5-kb upstream from the transcription start point. The same binding site was detected in ChIP-on-chip experiments using a line expressing Flag-tagged SCF and anti-Flag antibody (data not shown). Tiling array experiments showed that SCF is present in the Abd-B promoter. The occupancy of SCF at the same site in the Abd-B promoter was confirmed using conventional ChIP assays (data not shown).
Abd-B promoter activity is reduced by scf mutation
To test whether the scf1 mutation affects promoter activity of Abd-B, we performed transgenic reporter assays. Abd-Bpp spanning 4.1 kb of the 5' flanking region, 1.2 kb of mRNA leader and some coding sequence was used as a basal promoter of Abd-B in the reporter assays (Fig. 4A) (Busturia & Bienz 1993; Estrada et al. 2002). In two independent transgenic lines, expression of the reporter lacZ was reduced about 50% in scf1 GLC embryos compared with wild-type embryos at 5–16 h AEL (Fig. 4B). This result demonstrates that SCF positively regulates activity of the Abd-B promoter.
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Is the SCF-dependent activation of autosomal gene expression specific to Abd-B or applicable to other genes? To address the issue, we compared distributions of SCF and the Ser 5-phosphorylated form of RNA polymerase II on polytene chromosomes. This form represents a molecule engaged in early-stage transcriptional elongation (Phatnani & Greenleaf 2006). We identified co-localization of SCF with Ser 5-phosphorylated RNA polymerase II in many sites, including early ecdysone puffs (Fig. 5A–C). This result suggests that SCF occupies multiple transcriptionally active loci on polytene chromosomes.
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Discussion |
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Cooperation between scf and trxG in the regulation of Abd-B
RNAi knockdown of scf caused homeotic transformation related to loss of Abd-B function and synthetic lethality with an Abd-B mutation. This prompted us to investigate genetic interactions between scf and trans-regulators of Abd-B. We found that scf1 suppresses posterior transformation in Pc mutants and enhances anterior transformation in trxG mutants. These results suggest that scf functions together with trxG for the maintenance of Abd-B expression. As topoisomerase II, the partner of SCF for supercoiling activity, is present around Fab-7 (Lupo et al. 2001), we expected localization of SCF to regulatory regions of Abd-B such as the Fab-7 boundary or Pc response elements. However, tiling array experiments revealed occupancy immediately upstream of Abd-B. Some trxG proteins execute their function at Pc/trx response elements, but others regulate transcription at promoters or the beginning of coding sequences. For example, Brahma interacts with another trxG protein, Osa, to activate transcription from the Antennapedia P2 promoter (Vázquez et al. 1999) or the Abd-B promoter (Youhei Ogasawara, unpublished data). The TAC1 complex containing Trx predominantly localizes to the beginning of the hsp70 coding sequence to activate transcription (Smith et al. 2004). Similarly SCF may facilitate basal transcription of Abd-B together with trxG proteins.
SCF positively regulates promoters
Tiling array experiments revealed occupancy of SCF around 1.5-kb upstream of the transcription start site of Abd-B. This is consistent with the idea that SCF directly activates Abd-B transcription through the SCF site. However, SCF is not a sequence-specific DNA-binding protein and we were thus unable to narrow down the SCF site at a nucleotide level. As deletion or base substitutions of ~300 bp (i.e. average size of the DNA fragments in tiling array experiments) could influence not only SCF occupancy, but also binding of some trans-regulators of Abd-B, excluding the possibility that SCF indirectly activates Abd-B transcription through control of the expression of putative trans-regulators is difficult using transgenic reporter assays with deletion or base-substitution derivatives. Instead, we employed a recently developed statistical approach to identify correlations between SCF and promoters. The results of immunostaining on polytene chromosomes and ChIP-on-chip analyses covering an 11-Mbp region of the third chromosome suggest that SCF preferentially occupies around the transcriptional start sites of many active genes. This supports the idea that SCF directly activates promoters of multiple genes.
Mechanism of SCF action
How does SCF enhance promoter activity? Several possibilities exist for the actions of SCF. First, negative supercoils of DNA produced by SCF and topoisomerase II can facilitate binding of TFIID to promoters and activate transcription (Mizutani et al. 1991; Tabuchi et al. 1993). Second, negative supercoiling can also facilitate TFIIH-dependent unwinding of DNA to start transcription (Parvin & Sharp 1993). Third, as SWI/SNF-type chromatin remodeling factors introduce negative supercoils into DNA, SCF-dependent negative supercoiling can affect chromatin remodeling (Havas et al. 2000; Lia et al. 2006). Notably, Furuhashi et al. (2006) proposed that SCF-dependent supercoiling forms and/or maintains transcriptionally active open chromatin on the male X chromosome by counteracting ISWI action toward a more condensed chromatin state. Finally, SCF may activate promoters through an unknown mechanism independent of DNA supercoiling. Further studies are necessary to clarify this issue.
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Experimental procedures |
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Flies were raised on standard agar/cornmeal/yeast medium. RNAi knockdown of scf was performed as reported previously (Furuhashi et al. 2006). J2.4 was a gift from Julian Ng and L. Luo (Ng et al. 2002). Genomic scf rescue construct (Gscf) and Abd-Bpp (Busturia & Bienz 1993; Estrada et al. 2002) transgenic flies were produced by P-element-mediated transformation using the Df(1)w67c23, y1 (yw) strain as a host. Abd-Bpp vector was a gift from A. Busturia. Pc3 (Lewis 1978; Duncan 1982), P[ovoD1]3L P[FRT]2A (Chou et al. 1993), Gl P[FRT]2A and TTG balancer (Halfon et al. 2002) were obtained from the Bloomington Stock Center (Bloomington, IN). scf1 P[FRT]2A was obtained by crossing over in scf1/Gl P[FRT]2A female. GLC embryos were prepared as described previously (Chou et al. 1993). Both trxB11 and RpII140z43 were gifts from J. Tamkun.
Quantitative RT-PCR
Quantitative RT-PCR was performed as reported previously (Furuhashi et al. 2006) with some modifications. Synthesis of cDNA was performed using an ExScript RT reagent Kit (Takara, Kyoto, Japan). Quantitative PCR was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara) and the following gene-specific primers: Abd-B (Abd-B-RB, Abd-B(m)), 5'-ATCAAAAACAAACGCCAACC-3' and 5'-TCAGTTTTCATTCGGTCAATCC-3'; lac-Z, 5'-CGCCAGTCAGGCTTTCTTTCAC-3' and 5'-CAATGCGGGTCGCTTCACTTAC-3'; Act5C, 5'-CCCTCGTTCTTGGGAATGG-3' and 5'-CGGTGTTGGCATACAGATCCT-3'; β1-tubulin and Rp49 as described (Furuhashi et al. 2006).
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed essentially as described previously (Schwartz et al. 2003), with the following modifications. Embryos were collected 5–16 h AEL. Embryos (1 g wet weight) were homogenized in a prechilled dounce homogenizer with 10 mL of ice-cold buffer A (0.3 M sucrose, 2 mM MgOAc, 3 mM CaCl2, 10 mM Tris–HCl (pH 8), 0.3% Triton X-100, 0.5 mM dithiothreitol) and the homogenate was filtered through two layers of nylon mesh. Exactly 1 volume of cross-linking buffer [0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Tris–HCl (pH 8)] at 40 °C was added to bring the temperature to 20 °C. Formaldehyde (36% formalin solution) was immediately added to a final concentration of 0.36% and swirled gently for 1 min. Then, 2.5 M glycine was added to a final concentration of 125 mM to stop the reaction. The mixture was spun down for 3 min in a clinical centrifuge at 2110 g, and the supernatant was discarded. The pellet was resuspended in 6 mL of a sonication buffer (10 mM Tris–HCl, 1 mM EDTA, 0.5 mM EGTA) and 1/100 volume of protease inhibitor cocktail (SIGMA, St. Louis, MO) was added. This solution was sonicated on ice in a Branson digital sonifier for a total of 8 min. Each burst of the sonifier lasted 30 s and was followed by a 1-min cool off. Sonicated material was spun down at 20 000 g. Buffer ingredients of the supernatant were adjusted to RIPA (140 mM NaCl, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.3% SDS, 0.1% sodium deoxycholate). Solution was gently mixed for 10 min followed by centrifugation at 10 000 g for 5 min. A 300-µL portion of supernatant was pre-cleared by incubating with 50 µL of protein A agarose (Upstate Technology, Billerica, MA) at 4 °C for 30 min followed by spin down. Next, 700 µL of RIPA were added to supernatant supplemented with 1/100 volume of protease inhibitor cocktail (SIGMA). The solution was mixed with 50 µL of Salmon Sperm DNA/Protein A Agarose (Upstate Technology) and 100 µL of anti-SCF serum (
CT of Kobayashi et al. 1998) followed by rotation at 4 °C overnight. The agarose was washed 4 times with RIPA, once with a RIPA high-salt buffer (RIPA containing 500 mM NaCl), once with a LiCl wash buffer (250 mM LiCl, 10 mM Tris–HCl at pH 8.0, 1 mM EDTA at pH 8.0, 0.5% sodium deoxycholate, 0.5% NP-40), and finally twice with TE (10 mM Tris–HCl at pH 8.0, 1 mM EDTA at pH 8.0). Each wash step was performed at 4 °C for 5 min. After washing, beads were resuspended in 200 µL of a reverse cross-link buffer (300 mM NaCl, 0.5% SDS in TE) and incubated overnight at 65 °C. Supernatant was transferred to a new tube and the agarose was re-extracted with TE. Combined supernatant (400 µL) was treated with RNaseA (0.1 mg/mL) at 37 °C for 2 h, and then with Proteinase K (0.1 mg/mL) at 55 °C for 30 min. DNA was recovered by phenol–chloroform extraction followed by ethanol precipitation and resuspended in 70 µL of 10 mM Tris–HCl (pH 8.0). Input chromatin DNA was also reverse cross-linked and purified for reference.
DNA labeling
To perform hybridization using a microarray, purified DNA was labeled essentially as reported previously (Boyer et al. 2005) with the following modifications. DNA was blunted and ligated to linker and amplified using two-stage PCR. Amplified DNA was labeled with a CGH Labeling kit (Invitrogen, Carlsbad, CA) and purified with a CGH column (Invitrogen). Immunoenriched DNA was labeled with Cy5 fluorophore, while input chromatin DNA was labeled with Cy3 fluorophore. Each 0.5 µg portion of labeled DNA was combined and hybridized to arrays in Agilent hybridization chambers for 16 h at 65 °C. Arrays were then washed and scanned using an Agilent microarray scanner.
DNA microarray analysis
DNA microarray analysis was performed as described previously (Boyer et al. 2005). We used Agilent-013844 Drosophila ChIP-on-Chip Set 44K, Array 8 (G4475A), which is described in GEO (Accession number GPL4536 [NCBI GEO] ). A whole-chip error model was used to calculate confidence values from the enrichment ratio and the signal intensity of each probe (probe P-value) and that of each set of three neighboring probes (probe-set P-value). These processes were performed according to Feature Extraction and ChIP analytics (Agilent). Probe-sets with significant probe-set P-values (P < 0.001) and significant individual probe P-values were judged to be bound as described previously (Boyer et al. 2005). Raw and processed microarray data are available from the Center for Information Biology Gene Expression Database with the accession number CBX25.
The dataset of SCF binding profiles was compared with the dataset of transcription start sites obtained from DroSpeGe Mart <http://insects.eugenes.org/BioMart/martview>. Data mining and visualizing were performed using Excel software (Microsoft, Redmond, NJ).
Immunostaining of polytene chromosomes
Immunostaining of polytene chromosomes was performed as reported previously (Furuhashi et al. 2006). Polytene chromosomes were stained with purified antibodies against SCF (1:25) and monoclonal antibody H14 (1 : 100; Covance, Princeton, NJ), followed by anti-rabbit Alexa488 (1 : 500, Molecular Probes, Carlsbad, CA) and anti-mouse IgM Cy3 (1 : 500, Jackson Immuno Research, West Grove, PA).
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Biology, O. Wayne Rollins Research Center, Emory University, Atlanta, GA 30322, USA.
* Correspondence: E-mail: shirose{at}lab.nig.ac.jp |
References |
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Accepted: 12 September 2007
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