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First published online 29 August 2006
doi: 10.1242/jcs.03150
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Research Article |
Department of Developmental Genetics and Molecular Physiology, Institute of Biotechnology, National Autonomous University of México, Av. Universidad 2001, Cuernavaca Morelos 62250, Mexico
* Author for correspondence (e-mail: marioz{at}ibt.unam.mx)
Accepted 22 June 2006
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: TFIIH, Drosophila, Early embryo, Dynamics
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Introduction |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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). XPD is component of both complexes and it seems to be the bridge between the core and the CAK (Egly, 2001). TFIIH has several enzymatic activities: XPB and XPD are DNA helicases, CDK7 is a kinase and the p44 subunit is a ubiquitin ligase (Takagi et al., 2005). Mutations in either of the genes encoding XPB or XPD can produce three hereditary human disorders known as Xeroderma pigmentosum (XP), patients with combined symptoms of Xeroderma pigmentosum and Cockayne syndrome (XP/CS) and Trichothiodystrophy (TTD) (Lehmann, 1998; de Boer and Hoeijmakers, 1999; Nance and Berry, 1992; Cleaver, 2000; Rolig and McKinnon, 2000; Lehmann, 2002). It has recently been reported that mutations in the new TFIIH subunit p8 also produce TTD (Giglia-Mari et al., 2004).
Besides its dual role in transcription, TFIIH also has a central role in the mechanism of nucleotide-excision repair (NER) (Egly, 2001; Zurita and Merino, 2003), and there is increasing evidence that the CAK complex is involved in cell-cycle regulation (Chen et al., 2003; Fisher, 2005). In transcription, TFIIH promotes the formation of the open complex and it is generally accepted that its kinase activity targets the CTD domain of the RNA pol II, in particular the phosphorylation of Ser5 in the heptapeptide repeat (Lu et al., 1992; Rossignol et al., 1997). These events facilitate the escape of the RNA pol II from the promoter. Interestingly, in vitro experiments have shown that in some promoters RNA pol II transcription can be achieved without TFIIH kinase activity (Mäkelä et al., 1995). Another report shows that the conditional ablation of the MAT1 subunit in Schwann cells does not affect transcription (Korsisaari et al., 2002). By contrast, CDK7 is required for the transcription of the Drosophila heat shock genes and RNA pol II phosphorylation in third-instar larvae salivary glands (Schwartz et al., 2003). Also, the development of structures that require high levels of transcription are affected in a Cdk7 mutant (Merino et al., 2002).
In Drosophila, a Cdk7 dominant-negative mutant was found to delay embryonic transcription (Leclerc et al., 2000). In addition, CDK7 is required for both mRNA transcription and cell-cycle progression in early Caenorhabditis elegans embryos (Wallenfang and Seydoux, 2002). Therefore, the role of the CAK complex during development is still not well understood.
In many animals, the early embryo does not require the synthesis of RNA. Activation of transcription generally occurs at the mid-blastula transition and is an essential requirement for subsequent developmental stages (Davidson, 1986). In Drosophila, early embryo development passes through a syncytial stage, in which 13 synchronic nuclear divisions occur without cell division. In addition, early mitotic divisions occur with partial nuclear envelope breakdown (Stafstrom and Staehelin, 1984). No transcription occurs during these synchronic nuclear divisions, with the exception of the cycle elongation in division 10, in which histone, Gap and pair rule genes begin to be transcribed. During cellularization, global transcription is activated and there is an increase in phosphorylation of the RNA pol II C-terminal domain (CTD) (Seydoux and Dunn, 1997). Therefore, early fly embryo development is an excellent model to analyze the intracellular traffic and dynamics of the basal transcription machinery at transcription onset at the mid-blastula transition (MBT).
In this work, we analyzed the intracellular dynamics of the TFIIH core and CAK complexes in the early Drosophila embryo at the MBT. Our results demonstrate that most of the CAK and the core complexes follow different dynamics and subcellular distribution. However, both complexes seem to be involved in gene activation at the onset of transcription. We also demonstrate that the translocation of XPB and XPD into the nucleus at this stage is inhibited by the inactivation of any subunit of the TFIIH core and that this affects transcription activation. These results are the first to address the assembly and trafficking of a transcriptional complex at the onset of zygotic transcription at MBT.
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Results |
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) distribution at different stages of embryo development. XPB protein is initially deposited in the early syncytial blastoderm by maternal contribution and is located in the cytoplasm (Fig. 1a). By nuclear division eight, nuclei migrate to the embryo periphery while XPB also concentrates in the embryo border region while remaining cytoplasmic (Fig. 1b). Interestingly, at this stage XPB surrounds the nuclei in cytoplasmic domains or energids that delimitate the space that each cell will occupy in the cellular blastoderm (Fig. 1c,c'). By nuclear division ten, part of the XPB signal is located inside the nuclei at the embryo periphery (Fig. 1d,g). At this stage syncytial nuclei have a prolongation of their replication phase that allows the transcription of the first zygotic genes. By cellularization at nuclear division 13-14, most XPB signal is located inside the nucleus (Fig. 1e,h). At this stage RNA pol II transcription is activated in the early Drosophila embryo. During gastrulation, XPB is preferentially maintained inside the interphase nuclei while it is clearly excluded from mitotic chromosomes (Fig. 1f,i). XPD displays identical dynamics to XPB, and both proteins colocalize throughout development (supplementary material Figs S1, S2).
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Since it has been reported that XPD anchors the CAK complex with TFIIH, we performed simultaneous immunostaining with XPD and CDK7. At the resolution of the confocal microscope, the two proteins appear not to colocalize (Fig. 3). Western blot experiments with cytoplasmic and nuclear fractions from cellularized embryos confirmed that most of the XPD signal is nuclear and that CDK7 is preferentially located in the cytoplasm (supplementary material Figs S1, S2). These data suggest that most of the core and CAK complexes do not interact during early Drosophila embryo development.
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). Therefore, we decided to analyze the subcellular distribution of the two TFIIH subcomplexes in terminally differentiated tissues such as third-instar salivary glands. We found that in differentiated tissues, both the CAK (CDK7 and MAT1) and the core (XPD and XPB) are preferentially nuclear (Fig. 4a,b; data not shown). In polytene chromosome preparations, CDK7 and XPB co-localize at most chromosomal sites (Fig. 4c). However, we identified some regions where only CDK7, but not XPB or XPD is detectable (Fig. 4c enlarged image). Similar results were observed with chromosome immunostaining against MAT1 and XPD (Fig. 4d). These results show that, in contrast to the CAK cellular distribution in the Drosophila embryo, at larval stages this complex is preferentially nuclear and it co-localizes in most, but not all chromosomal sites with the TFIIH core subunits.
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The CAK and the core subcomplexes of TFIIH are positioned in gene promoters activated at the onset of transcription in the early Drosophila embryo
The immunolocalization experiments in early Drosophila embryos suggest that most of the CAK is in the cytoplasm and that it may not be involved in transcription or that it has a limiting role at these stages. However, confocal observations and western blots against different cellular fractions may not be sensitive enough to distinguish a small amount of CAK that might be localized on the chromatin. On the other hand, it has been reported that the overexpression of a dominant-negative form of cdk7 delays fushi taratzu (ftz) gene transcription (Leclerc et al., 2000). Although the authors argue that this is a clear transcriptional defect, a problem in the cell cycle cannot be ruled out. Defects in the cell cycle caused by Cdk7 mutations may affect the transition from synchronic nuclear division to asynchronous divisions and therefore delay the onset of transcription. Consequently, we decided to explore whether CAK participates in the transcription of genes that are activated between nuclear divisions 10-14. To achieve this, we performed chromatin immunoprecipitations (ChIP) experiments using antibodies against CDK7, XPD, XPB and RNA pol II. The selected targets were zygotic hunchback (hb) and histone H3 promoters. The zygotic hb and the H3 promoters are activated at nuclear division 10 elongation. As controls, we tested the promoter of the salivary-gland-specific gene Sgs5, which is not expressed in the embryo, the second exon of the hb gene and an exon of the Drosophila ATRX homologue, which is not expressed at these embryonic stages (our unpublished results).
ChIP experiments show that CDK7, XPD, XPB and RNA pol II are positioned in hb, and H3 promoters (Fig. 5). As expected, RNA pol II, but not CDK7, XPB and XPD, can be found at the hb exon. In addition, XPB, XPD and CDK7 are absent from the Sgs5 promoter. In the ATRX exon none of these proteins were identified. These results show that even if CAK is not visualized by immunostaining the nuclei of early Drosophila embryos and that most of it is in the cytoplasm, some CAK is positioned at the promoter of genes that are being transcribed at these developmental stages, supporting a role of CAK in early Drosophila embryo transcription.
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To determine if the CAK and the core complexes subunits interact in the early fly embryo cytoplasm, we performed co-immunoprecipitations (CoIP) of 1-2.5 hour embryonic cellular and nuclear fractions. Anti-CDK7 and anti-XPB antibodies were used for CoIP experiments. Precipitated samples were analyzed for the presence of CDK7, MAT1, XPB and XPD by western blotting. After CoIP of the cytoplasmic fraction with anti-CDK7 antibody, MAT1, XPD and CDK7 can be detected in a western blot, forming the XPD-CAK transitory complex with CycH (Fig. 6). XPB does not co-immunoprecipitate with CDK7 (Fig. 6), suggesting that even though all the components of TFIIH are present in the early Drosophila embryo cytoplasm, the complete assembled TFIIH, with all its ten subunits, is not present in this cellular fraction. On the other hand, in CoIP experiments of the cytoplasmic fraction using the anti-XPB antibody, XPD can be identified in western blots of CoIP material, but not CDK7 and MAT1 (Fig. 6). Complementary experiments with the CDK7 antibody using a nuclear fraction enriched with transcriptionally active chromatin (Kamakaka and Kadonaga, 1994), we found that CDK7, MAT1, XPD and XPB can be identified, suggesting that the CAK and the core are assembled in the nucleus thus forming the TFIIH ten-subunit factor (Fig. 6). CoIP of this fraction with the anti-XPB antibody pulls down XPD together with trace amounts of CDK7 and MAT1 (Fig. 6), indicating that most of the core complex is not interacting with the CAK, in agreement with the confocal observations.
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XPB and XPD are assembled in the core of TFIIH to entry into the nucleus at the mid-blastula transition and are required for the onset of transcription
CoIP experiments with nuclear and cytoplasmic fractions from early embryos show that the CAK complex interacts with XPD in the cytoplasm, forming a transitory complex, and that this complex is probably transported inside the nucleus at the onset of transcription. These data also suggest that the core is assembled in the cytoplasm and transported to the nuclei. To determine if the core has to be assembled in the cytoplasm to enter the nuclei at transcription activation, we neutralized either XPB or XPD proteins in syncytial blastoderm embryos by injecting the corresponding antibodies. After injection, we looked for the localization of these two core components in cellularized and gastrulated embryos. We microinjected embryos 0-30 minutes old with the antibodies, fixed them, allowed to develop for 2 hours and stained them with either anti-XPB (in embryos injected with the XPD antibody), or anti-XPD (in embryos injected with the XPB antibody). The rationale for this experiment is that if both proteins are required to form the core to enter the nucleus, the neutralization of either one will interfere with the traffic of the other from the cytoplasm into the nucleus. As a control, we microinjected a GFP antibody, which does not recognize any protein in Drosophila. We also injected an antibody against CDK7 and stained for XPB and XPD. As control for an independent nuclear factor, we also stained the injected embryos with an anti-TBP antibody.
The results presented in Fig. 7 show that the neutralization of the XPD protein reduces the quantity of XPB inside of the nucleus, which remains preferentially cytoplasmic in embryos initiating gastrulation, a stage in which we have demonstrated that XPB and XPD are preferentially nuclear (Fig. 7). When we injected the XPB antibody, XPD preferentially remains cytoplasmic (Fig. 7). On the other hand the control antibody did not affect the nuclear localization of either XPB or XPD in cellular blastoderm and gastrulated embryos (Fig. 7). Neither XPB nor XPD antibodies affect TBP nuclear localization, indicating that the effect is specific for TFIIH (Fig. 7). We were not able to visualize what happened when we neutralized CDK7, because the antibody immediately arrested development, producing aberrant mitosis, suggesting a role for CDK7 in the regulation of the first mitotic cycles of the early Drosophila embryo (Fig. 7). This observation disagrees with the previous report by Leclerc et al. (Leclerc et al., 2000) in which the expression of a Cdk7 dominant-negative mutant of Cdk7 does not alter the timing of the first 13 embryonic nuclear cycles. However, differences in our work may be due to the use of different strategies and conditional cdk7 mutant versus inactivation of Cdk7 by antibodies.
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To determine if the inactivation of XPB or XPD affects transcription at the mid-blastula transition, we analyzed the expression of the fushi taratzu (ftz) gene by in situ hybridization in embryos that were microinjected with the XPB or XPD antibodies. In the early fly embryo, ftz is one of the first zygotic transcripts to be expressed and we found that the ftz expression pattern was affected in cellularized and gastrulated embryos injected with XPB and XPD antibodies (Fig. 8). These results show that XPB and XPD and are required for the onset of transcription at the mid-blastula transition.
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Discussion |
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The CAK complex phosphorylates Cdks that are fundamental for cell-cycle progression (Morgan, 1995). It has been reported that the activation of the Cdc2/Cyclin A and Cdc2/Cyclin B complexes in Drosophila requires Cdk7 (Larochelle et al., 1998; Larochelle et al., 2001). Cyclin A and B, but not Cdc2, levels fluctuate during these developmental stages (Foe et al., 1993). As a large proportion of Cdc2 is in the cytoplasm, it is possible that the main function of CAK in the cytoplasm may be the regulation of Cdc2 activity. In relation to this, it has been recently proposed that XPD levels regulate CDK7 activity and are therefore central for the control of the cell cycle in the Drosophila embryo. In accordance with this, it has been reported that levels of XPD but not other TFIIH core components like XPB, drop during mitosis, suggesting a role for CDK7 in regulating the cell cycle (Chen et al., 2003). However, we observed that XPB and XPD have a similar expression pattern and co-localize at all the analyzed stages, and that their relative levels do not change during mitosis (supplementary material Figs S1, S2). In agreement with reports that show a major function of CDK7 in the M phase of the cell cycle (Larochelle et al., 1998), the inactivation experiments suggest a central role of CDK7 in the control of mitosis in the early blastoderm embryo (Fig. 7). In addition, the XPD antibody inactivation did not affect cell-cycle progression at these stages. On the other hand, we cannot discard a possible function of the CAK and core complexes in the cytoplasm beyond cell-cycle regulation. For instance, it is known that CAK also interacts with RNA, in particular CycH with the U1 snRNA and there are reports of that suggest a possible role of XPB in mRNA translation regulation in yeast (Kwek et al., 2002; Guylas and Donahoue, 1992).
In terminally differentiated tissues, the CAK and the core complexes become mostly nuclear. Why does this change in the subcellular distribution of the CAK complex occur? It is possible that the high levels of transcription that occur in the salivary glands cells require that most of CAK be preferentially nuclear and the regulation of other Cdks by the CAK complex may not be required.
In polytene chromosomes, the CAK and core complexes colocalize in most chromatin domains and are enriched in the puffs. This is to be expected because the two complexes form TFIIH, which is a component of the basal transcription machinery. However, there are some chromosomal regions where only the CAK complex is present, a surprising observation because its role in transcription has always been linked with TFIIH. This suggests that some loci may require the presence of CAK but not the other components of TFIIH to be transcribed. Alternatively, it is possible that CAK may be involved in functions other than the phosphorylation of the CTD domain of RNA pol II in the chromatin. Furthermore the chromosomal positions where CDK7 or MAT1 are located without the TFIIH core are the same in different chromosomal squashes, suggesting a specific unknown function. The future identification and characterization of the loci within these chromosomal positions will be useful to elucidate the role of CAK in the absence of TFIIH in the chromosomes.
The CAK and core complexes are translocated from the cytoplasm to the nucleus at the mid-blastula transition
Little is known about the transit of the basal transcription machinery from the cytoplasm to the nucleus at the onset of transcription in the early animal embryo. In the case of Drosophila it has been reported that RNA pol II is present in syncytial blastoderm nuclei between mitotic cycles eight to ten, and that significant phosphorylation of the CTD occurs at cellular blastoderm (Seydoux and Dunn, 1997). It has also been reported that the TATA-binding protein (TBP), a component of TFIID, enters the nuclei at mitotic division eight (Wang and Lindquist, 1998). Both RNA pol II and TFIID are composed of several subunits and it is not known if the assembly of these complexes occurs in the cytoplasm or in the nucleus. In addition, an interesting and elegant study has shown that transcription activation in the early fly embryo is a gradual and stochastic process mediated by the nucleus to cytoplasm ratio (Pritchard and Schubiger, 1998).
In this work we present evidence that in the cytoplasm of the early Drosophila embryo a transitory complex of CAK-XPD, which does not interact with XPB, is present in the cytoplasm. On the other hand XPB and XPD do interact in the cytoplasm of early Drosophila embryo. Since XPB and XPD do not physically interact in TFIIH, as this requires p52 and p44 to be assembled in the core of TFIIH (Schultz et al., 2000; Jawhari et al., 2002), we propose that the assembled core complex is present in the cytoplasm of syncytial and cellular blastoderm embryos, but that it does not interact with the CAK-XPD complex. Based on these observations, we also propose that the core complex enters the nucleus independently and then assembles with the CAK in active chromatin domains forming the ten-subunit TFIIH to activate transcription at the mid-blastula transition (Fig. 9). At the mid-blastula transition, most of the CAK complex remains cytoplasmic without interacting with XPD, probably having a function in cell-cycle control (Fig. 9). This model is supported by the fact that the antibody inactivation of either XPB or XPD in early embryos blocks the entry of the two proteins to the nucleus and the onset of transcription. Interestingly, XPD or XPB inactivation does not affect embryo development from syncitial blastoderm to gastrulation, suggesting that development at these stages does not require TFIIH.
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). However, our results suggest that in early fly development, XPD must be assembled with other TFIIH subunits to enter the nuclei. There are examples of transitory precursor complexes that are needed for a factor to enter the nucleus (Lehmann et al., 2002). We propose that this is the case for TFIIH at the onset of transcription in the early Drosophila embryo (Fig. 9).
Maternal RNA encoding some transcription factors that control the establishment of first embryonic coordinates are translated in the syncytial blastoderm and its protein products transported into the nuclei before the mitotic cycle 10 (Hegde and Stephenson, 1993). However, specific gene activation of transcription by these factors only occurs at the onset of transcription after mitotic division ten. The fact the RNA pol II, TBP and other transcription factors migrate into the nuclei before TFIIH suggest that entrance of TFIIH into the nucleus is a key regulatory element to activate transcription at the mid-blastula transition. It is important to remark that these statements agree with the proposed hypothesis of Almouzni and Wolffe (Almouzni and Wolffe, 1995), in which the temporal regulation of transcription during early embryogenesis in some organisms results in a deficiency in the activity of basal transcription activators to the MBT. In addition, our results are supported by the well-known fact that general transcription is inhibited during mitosis and also that in this process many transcription factors are excluded from chromatin (Akoulitchev and Reinberg, 1998; Long et al., 1998).
In conclusion, the CAK and core subcomplexes of TFIIH have differential dynamics in the early fly embryo. At the onset of transcription, the core components are nuclear and most of the CAK remains cytoplasmic. However, a small portion of CAK is positioned at actively transcribed chromatin, in particular at promoters that are activated at the onset of transcription in the early embryo. Later in development, both subcomplexes are preferentially nuclear and co-localize in many chromosomal regions, although we observed a small subset of regions where only the CAK is present. Both the transitory complex CAK-XPD and the core are present in the cytoplasm of the syncytial blastoderm, from where they migrate into the nucleus to form the TFIIH ten-subunit complex and participate together with other components of the basal transcription machinery to initiate transcription.
This study opens several interesting questions for future research. For example, what are the mechanisms that modulate the core and CAK transport between the cytoplasm and the nucleus at the onset of transcription? Why is the CAK preferentially located in the cytoplasm in proliferating cells in the embryo and nuclear in differentiated cells in larval stages? Can the CAK be involved in transcription without interacting with the rest of TFIIH? The eventual elucidation of these questions will be relevant for the understanding of the different functions in which TFIIH is involved.
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Immunofluorescence
Embryos were dechorionated in 50% bleach, fixed in formaldehyde-heptane and devitellinized with methanol. Embryos were permeabilized and blocked in PBST and 4% goat serum for 9 hours. Primary antibodies were added at 1:1000 dilution in TBST and incubated at 4°C overnight. The following primary antibodies were used: Haywire (XPB) (Merino et al., 2002), DmXPD (Reynaud et al., 1999), DmCDK7, human CDK7, human MAT1 and Drosophila TBP. After washing in TBST, secondary antibodies (Cy2- or Cy3-conjugated goat anti-rabbit or rat; Rockland) were added at a concentration of 1:1500 in TBST for 1 hour and embryos were washed in TBST. DNA was counterstained with Sytox Green. Co-staining of embryos using an anti-rat-DmXPD polyclonal antibody with anti-rabbit Cdk7 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-rat XPB (our own preparation) was performed simultaneously. Salivary glands from third-instar larvae were dissected and immunostained as previously described (Reynaud et al., 1999).
Immunolocalization of TFIIH on polytene chromosomes
Fixation and spreading of the chromosomes essentially followed the protocol reported by Engels et al. (Engels et al., 1986) with modifications reported by Reynaud et al. (Reynaud et al., 1999). Co-staining of polytene chromosomes using an anti-rat-Hay or DmXPD polyclonal antibody and an anti-rabbit-CDK7 or MAT1 was performed simultaneously.
Chromatin immunoprecipitation and co-immunoprecipitation from cytoplasmic and nuclear fractions
Embryos at 30-180 minutes of development (
1.0 g) were dechorionated in 3% NaOCl for 2-3 minutes at room temperature. Crosslinking was performed in 1.5 ml of crosslinking solution (PBS, 1.8% formaldehyde) for 15 minutes at room temperature. The crosslinking reaction was stopped by washing thoroughly with PBST (Orlando et al., 1997). Embryos were suspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), with protease inhibitors, and incubated for 10 minutes on ice. Chromatin was sonicated until DNA fragments between of 200 and 1000 base pairs length were obtained. The samples were centrifuged at 14,000 g for 10 minutes at 4°C. The supernatant was diluted ten times in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl) with protease inhibitors. Samples were taken, the crosslinking reverted and used for the input reaction. Pre-clearing was performed and 1 µg of the antibody was added: anti-DmCDK7 (Santa Cruz Biotechnology); anti-DmXPD (our own preparation); anti-XPB (our own preparation) or anti-RNA pol II-CTD domain (Covance) and the sample incubated for 3 hours at 4°C. As a negative control we used a chromatin extract without antibody. 30 µl of Sepharose-Protein G was then added for 1 hour at 4°C. The antibody-chromatin complex was washed first with high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), then with LiCl immune wash buffer and twice more with TE. The precipitated complex was suspended in TE and incubated with RNase for 30 minutes at 37°C. The sample was then incubated with 0.5% SDS and 0.5% Proteinase K for 1 hour at 45°C. DNA was extracted with phenol-chloroform several times and precipitated. DNA was used for radioactive PCR using specific oligonucleotides that amplify fragments of 400 bp for the targets indicated in the results and Fig. 5.
For co-immunoprecipitations (CoIP), cytoplasmic and nuclear fractions were prepared from embryos, about 1-3 g, collected after 1-2.5 hours of development as described in Reynaud et al. (Reynaud et al., 1997). The nuclear preparation was subfractioned to obtain transcriptionally active chromatin (Kamakaka and Kadonaga, 1994). In order to confirm the absence of cross-contamination of cytoplasmic and nuclear fractions, we performed western blots against superoxide dismutase (SOD) enzyme, which is strictly cytoplasmic in both fractions. CoIP was performed according to Leclerc et al. (Leclerc et al., 1996).
XPB and XPD inactivation by injection of specific antibodies and in situ hybridization
To obtain embryos at the correct stage of development (in syncytial blastoderm), flies were allowed to lay eggs on fresh agar trays for 30 minutes. These trays were then removed and embryos were dechorionated. Then embryos were aligned on double-sided sticky tape attached to microscope slides and desiccated for 10 minutes. Embryos were injected dorsally under halocarbon oil. We injected about 1000 embryos with anti-XPD, anti-XPB and anti-Cdk7 antibodies (80 µg/ml). Anti-XPB or -XPD injected antibodies were affinity purified against their respective specific epitopes. Embryos were also microinjected with a control antibody that was affinity-purified mouse anti-GFP polyclonal antibody. After injections, embryos were aged at 25°C for 2.5 hours, washed free of halocarbon oil with 100% heptane, fixed in 100% heptane, 37% formaldehyde (1:1) for 5 minutes. Embryos were then fixed and processed for immunofluorescence as described above. In situ hybridization in microinjected embryos was performed according the standard protocols reported by Mullen and DiNardo (Mullen and DiNardo, 1995).
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Footnotes |
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References |
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Akoulitchev, S. and Reinberg, D. (1998). The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7. Genes Dev. 15, 3541-3550.
Almouzni, G. and Wolffe, A. P. (1995). Constrains on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis. EMBO J. 8, 1752-1765.
Chen, J., Larochelle, S., Li, X. and Suter, B. (2003). Xpd/Ercc2 regulates CAK activity and mitotic progression. Nature 424, 228-232.[CrossRef][Medline]
Cleaver, J. E. (2000). Common pathways for ultraviolet skin carcinogenesis in the repair and replication defective groups Xeroderma pigmentosum. J. Dermatol. Sci. 23, 1-11.[CrossRef][Medline]
de Boer, J. and Hoeijmakers, J. H. J. (1999). Cancer from outside, aging from the inside: mouse models to study the consequences of defective nucleotide excision repair. Biochimie 81, 127-137.[Medline]
Davidson, E. H. (1986). Gene Activity in Early Development. Orlando Fl: Academic Press.
Egly, J. M. (2001). TFIIH: form transcription to clinic. FEBS Lett. 498, 124-128.[CrossRef][Medline]
Engels, W. R., Preston, C. R., Thompson, P. and Eggleston, W. B. (1986). In situ hybridization to Drosophila salivary gland chromosomes with biotinylated probes and alkaline phosphatase. BRL Focus 8, 6-8.
Fisher, R. P. (2005). Secrets of a double agent: CDK7 in cell cycle control and transcription. J. Cell Sci. 118, 5171-5180.
Foe, V. E., Odell, M. G. and Edgar, B. A. (1993). Mitosis and Morphogenesis in the Drosophila embryo: point and counterpoint. In The development of Drosophila melanogaster (ed. M. Bate and A. Martinez Arias) Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp: 149-300.
Giglia-Mari, G., Coin, F., Ranish, J. A., Hoogstraten, D., Theil, A., Wijgers, N., Jaspers, N. G., Raams, A., Argentini, M., van der Spek, P. J. et al. (2004). A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nat. Genet. 36, 714-719.[CrossRef][Medline]
Guylas, K. D. and Donahue, T. E. (1992). SSL2, a suppressor of a stem-loop mutation in the HIS4 leader encodes the yeast homologue of human ERCC-3. Cell 69, 1031-1042.[CrossRef][Medline]
Hegde, J. and Stephenson, E. C. (1993). Distribution of swallow protein in egg chambers and embryos of Drosophila melanogaster. Development 119, 457-470.[Abstract]
Jawhari, A., Laine, J. P., Dubaele, S., Lamour, V., Poterszman, A., Coin, F., Moras, D. and Egly, J. M. (2002). p52 Mediates XPB function within the transcription/repair factor TFIIH. J. Biol. Chem. 277, 31761-31767.
Kamakaka, R. T. and Kadonaga, J. T. (1994). The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos. Methods Cell Biol. 44, 225-235.[Medline]
Korsisaari, N., Rossi, D. J., Paetau, A., Charnay, P., Henkemeyer, M. and Makela, T. P. (2002). Conditional ablation of the Mat1 subunit of TFIIH in Schwann cells provides evidence that Mat1 is not required for general transcription. J. Cell Sci. 115, 4275-4284.
Kwek, K. Y., Murphy, S., Furger, A., Thomas, B., O'Gorman, W., Kimura, H., Proudfoot, N. J. and Akoulitchev, A. (2002). U1 snRNA associates with TFIIH and regulates transcriptional initiation. Nat. Struct. Biol. 9, 800-805.[Medline]
Larochelle, S., Pandur, J., Fisher, R. P., Salz, H. and Suter, B. (1998). Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes Dev. 12, 370-381.
Larochelle, S., Chen, J., Knights, R., Pandur, J., Morcillo, P., Erdjument-Bromage, H., Tempst, P., Suter, B. and Fisher, R. P. (2001). T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity. EMBO J. 20, 3749-3759.[CrossRef][Medline]
Leclerc, V., Tassan, J. P., O'Farrell, P. H., Nigg, E. A. and Leopold, P. (1996). Drosophila Cdk8, a kinase partner of cyclin C that interacts with large subunit of RNA polymerase II. Mol. Biol. Cell 7, 505-513.[Abstract]
Leclerc, V., Raisin, S. and Leopold, P. (2000). Dominant-negative mutants reveal a role for the Cdk7 kinase at the mid-blastula transition in Drosophila embryos. EMBO J. 19, 1567-1575.[CrossRef][Medline]
Lehmann, A. R. (1998). Dual functions of DNA repair genes: molecular, cellular and clinical implications. BioEssays 20, 146-155.[CrossRef][Medline]
Lehmann, A. R. (2002). Ageing: repair and transcription keep us from premature ageing. Curr. Biol. 12, 550-551.[CrossRef]
Lehmann, A., Janek, K., Braun, B., Kloetzel, P. M. and Enekel, C. (2002). 20 S proteosomes are imported as precursor complexes into the nucleus of yest. J. Mol. Biol. 317, 401-413.[CrossRef][Medline]
Long, J., Leresche, A., Kriwacki. and Gottesfeld, R. W. (1998). Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis. Mol. Cell. Biol. 18, 1467-1476.
Lu, H., Zawel, L., Fisher, L., Egly, J. M. and Reinberg, D. (1992). Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II. Nature 358, 641-645.[CrossRef][Medline]
Mäkelä, T. P., Parvin, J. D., Kim, J., Huber, L. J., Sharp, P. A. and Weinberg, R. A. (1995). A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription. Proc. Natl. Acad. Sci. USA 92, 5174-5178.
Merino, C., Reynaud, E., Vazquez, M. and Zurita, M. (2002). DNA repair and transcriptional effects of mutations in TFIIH in Drosophila development. Mol. Biol. Cell 13, 3246-3256.
Morgan, D. O. (1995). Principles of CDK regulation. Nature 374, 131-134.[CrossRef][Medline]
Mounkes, L. C., Jones, R. S., Bee-Choo, L., Gelbart, W. and Fuller, M. T. (1992). A Drosophila model for Xeroderma pigmentosum and Cockayne's syndrome: haywire encodes the fly homologue of ERCC3, a human excision repair gene. Cell 71, 925-937.[CrossRef][Medline]
Mullen, J. R. and DiNardo, S. (1995). Establishing parasegments in Drosophila embryos: roles of the odd-skipped and naked genes. Dev. Biol. 169, 295-308.[CrossRef][Medline]
Nance, M. A. and Berry, S. A. (1992). Cockayne Syndrome: review of 140 cases. Am. J. Med. Genet. 42, 68-84.[CrossRef][Medline]
Orlando, V., Strutt, H. and Paro, R. (1997). Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205-214.[CrossRef][Medline]
Pritchard, D. K. and Schubiger, G. (1996). Activation of transcription in Drosophila embryos is a gradual process mediated by the nucleocytoplasmic ratio. Genes Dev. 10, 1131-1142.
Reynaud, E., Barajas, V., Bolsakov, S., Kafatos, F. C. and Zurita, M. (1997). The antisense suppression of the putative ribosomal protein S3a disrupts ovarian development in Drosophila melanogaster. Mol. Gen. Genet. 256, 462-467.[CrossRef][Medline]
Reynaud, E., Lomeli, H., Vazquez, M. and Zurita, M. (1999). The Drosophila melanogaster homologue of the Xeroderma pigmentosum D gene product is located in euchromatic regions and has a dynamic response to UV light-induced lesions in polytene chromosomes. Mol. Biol. Cell 10, 1191-1203.
Rolig, R. L. and McKinnon, P. J. (2000). Linking DNA damage and neurodegeneration. Trends Neurosci. 23, 417-423.[CrossRef][Medline]
Rossignol, M., Kolb-Cheynel, I. and Egly, J. M. (1997). Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J. 16, 1628-1637.[CrossRef][Medline]
Santagati, F., Botta, E., Stefanini, M. and Pedrine, M. A. (2001). Different dynamics of entry subunits of the repair/transcription factor TFIIH. Nucleic Acids Res. 29, 1574-1581.
Schultz, P., Fribourg, S., Poterszman, A., Mallouh, V., Moras, D. and Egly, J. M. (2000). Molecular structure of human TFIIH. Cell 102, 599-607.[CrossRef][Medline]
Schwartz, B. E., Larochelle, S., Suter, B. and Lis, J. T. (2003). Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo. Mol. Cell. Biol. 23, 6876-6886.
Seydoux, G. and Dunn, M. A. (1997). Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development 124, 2191-2201.[Abstract]
Stafstrom, J. P. and Staehelin, L. A. (1984). Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the Drosophila embryo. Eur. J. Cell Biol. 34, 179-189.[Medline]
Takagi, Y., Masuda, C. A., Chang, W.-H., Komori, H., Wang, D., Hunter, T., Joazeiro, C. A. P. and Kornberg, R. D. (2005). Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage. Mol. Cell 18, 237-243.[CrossRef][Medline]
Wallenfang, M. R. and Seydoux, G. (2002). cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos. Proc. Natl. Acad. Sci. USA 99, 5527-5532.
Wang, Z. and Lindquist, S. (1998). Developmentally regulated nuclear transport of transcription factors in Drosophila embryos enable the heat shock response. Development 125, 4841-4850.[Abstract]
Zurita, M. and Merino, C. (2003). The transcriptional complexity of the TFIIH complex. Trends Genet. 19, 578-584.[CrossRef][Medline]
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