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1 Department of Developmental Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan
2 Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan
3 CREST, Japan Science and Technology Agency (JST), Yata 1111, Mishima, Shizuoka 411-8540, Japan
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Abstract |
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Introduction |
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The subunit requirements for various individual functions of TFIIH have not been clearly elucidated. Biochemical studies have shown that 10 subunits which have been associated with TFIIH can be subdivided in two sub-complexes: core TFIIH and CAK. Core TFIIH consists of six subunits: p44, XPB, p62, p52, p34 and p8; and CAK is composed of three subunits: Mat1, cdk7 and cyclin H (Giglia-Mari et al. 2004). XPD is found in both sub-complexes, suggesting that XPD bridges CAK and core TFIIH (Egly 2001). Because each subcomplex contains at least one enzymatic subunit, it is possible that certain TFIIH functions are carried out by specific subcomplexes, rather than by the TFIIH holocomplex. Indeed, previous experiments suggest that while DNA repair requires core TFIIH, CAK is sufficient for cell cycle regulation (Rossignol et al. 1997; Yankulov & Bentley 1997; Tirode et al. 1999; Araújo et al. 2000; Vermeulen et al. 2000; Fisher 2005). These in vitro experiments, however, do not demonstrate that these subcomplexes are sufficient to perform TFIIH function in vivo, where TFIIH must integrate physiological information from the environment.
One method to determine what function is associated with a particular complex or subcomplex is to examine the consequences of mutations in "structural" subunits of TFIIH (p8, p52, p44, p62 and p34, Tirode et al. 1999; Giglia-Mari et al. 2004). Mutation of a single "structural" subunit can change the stability and function of the TFIIH complex that contains the subunit (Seroz et al. 2000; Tremeau-Bravard et al. 2001; Giglia-Mari et al. 2004). One such "structural" subunit that is strongly conserved throughout evolution is p52, a subunit of core TFIIH (Feaver et al. 1997; Marinoni et al. 1997; Lanning & Lafuse 1999). p52 has been proposed to mediate the binding of the XPB helicase/ATPase to other components of the TFIIH core complex (Jawhari et al. 2002). Loss of the p52 subunit is expected to affect functions performed by core TFIIH and the holocomplex, but not those that can be carried out by CAK subcomplex alone. Here, we present a functional analysis of the p52 subunit of TFIIIH in the Drosophila eye imaginal disc, a developing tissue where cell proliferation and cell fate determination are linked and occur simultaneously (Baker 2001).
The Drosophila eye imaginal disc is an excellent system to examine various functions of TFIIH, because cell proliferation and cell fate determination are under spatiotemporal control and cells at varying stages of development can be identified based on their position along the anterior–posterior axis of the disc epithelium (Baker 2001). Cells divide asynchronously until the third instar larva (the first mitotic wave), but then arrest prior to differentiation at the G1 stage in a strip of cells called the morphogenetic furrow. Posterior to the morphogenetic furrow, a minority of cells enter a differentiation program to become specific photoreceptor neurons, whereas the remaining majority of cells undergo synchronous cell division to produce a pool of uncommitted cells (the second mitotic wave, Baker 2001). Thus, all cells in this region must choose either to initiate photoreceptor differentiation or to re-enter a final mitosis. These features make the eye imaginal disc an ideal system for performing genetic analyses of p52 to unravel its role in TFIIH functions.
We assayed various TFIIH-dependent cellular functions in a clonal population of cells, where p52 levels were reduced. p52 mutant cells were capable of expressing various differentiation and cell fate markers, but had defects in DNA repair, and progression through a developmentally-regulated cell cycle event. Furthermore, we determined that both p52 and Cdk7 are required for the G1/S transition, a first demonstration in multicellular organisms. Cell cycle defects similar to that observed in our p52 mutants were also observed in mutants of another TFIIH core subunit, XPB. Our data indicate that the integrity of the TFIIH core is required to regulate cell cycle progression, implying that regulation of the cell cycle by TFIIH requires an intact holocomplex.
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Results |
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We generated loss-of-function mutations in the gene encoding p52 using line ep(3)0572, in which a P-transposable element is inserted 5 bp downstream of the translation initiation site of the p52 open reading frame (Fig. 1A). Excision lines were generated from which we identified line ex9-2, in which 989 bp of the genomic DNA (position +6 to +994, where +1 is the first nucleotide of the translation initiation codon) were deleted and replaced with 24 bp of unrelated sequence. These result in an early in-frame stop codon and suggests that line ex9-2 is likely to contain a null allele of p52. We also found that mutations in a previously identified locus, marionnette (mrn, Fuller 1986), fail to complement the lethality of ex9-2. Examination of the p52 genomic DNA of six of these mrn alleles revealed that all six alleles had changes within the p52 coding region: mrn2, mrn3, mrn4 and mrn5 had nonsense mutations, mrn6 contained a 207 bp deletion including the translation initiation codon, and mrn1 carried two mis-sense mutations within the open reading frame (Fig. 1A). Thus, the p52 subunit of TFIIH is encoded by the mrn locus, and we will refer to ex9-2 as mrn9. While this work was in preparation Fregoso et al. (2007) also reported that the mrn gene encodes the p52 subunit. In contrast to their report, we found two mis-sense mutations in mrn1: A318P and E340K.
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mrn is highly expressed in proliferating cells
We next characterized the expression pattern of mrn during development by in situ hybridization (Fig. 1B–G). Early embryos contain maternally supplied mrn mRNA. As development proceeds, expression becomes more uneven and is highest in the proliferating cells of the nervous system, Malpighian tubules and midgut (Fig. 1D,E). At larval stages, mrn is highly expressed in restricted regions of the imaginal discs, including the wing pouch in the wing imaginal disc, and the morphogenetic furrow in the eye imaginal disc, an indentation in the disc epithelium that acts as a moving front of differentiation (Fig. 1F,G). During eye development, cells become arrested in G1 within the morphogenetic furrow prior to differentiation (Baker 2001). The high expression levels of mrn in the morphogenetic furrow suggest that the requirement for p52/MRN is particularly high in this region.
mrn is required for cell proliferation in the developing eye
Because mrn mutant animals die before the function of the p52 subunit during eye development can be analyzed, we generated homozygous somatic clones of mrn9 and mrn1 alleles in otherwise heterozygous animals. Mitotic recombination was induced by the FRT/FLP technique using ey-FLP which targets the recombination event that generates a homozygous mutant founder cell to the eye imaginal disc (Fig. 2A–F). Proliferation of this founder cell produces a clonal population of mutant cells, which cannot produce MRN mRNA. Therefore, p52 subunit present in the mrn+/– mother cell should be serially diluted at each cell division.
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Transcription and translation can occur in mrn mutant clones
The defects in cell proliferation that we observed in mrn mutant clones could be a secondary consequence of defective transcription. To test this possibility, we first checked transcription in mrn mutant clones generated in the eye using a lacZ reporter gene driven by transcription factor Glass. Glass is expressed in all cells within the eye morphogenetic field where cell fate determination and differentiation is taking place (Ellis et al. 1993). In the third larval instar eye imaginal disc this reporter gene was expressed normally within mrn mutant clones (Fig. 3A–C). Furthermore, multiple markers for differentiation of specific cell types could be detected within mrn mutant clones (Figs 3D–O and 6A–C, G–I). These results suggest that even under conditions where a core p52 subunit of TFIIH is severely reduced (and possibly not present), cells can still transcribe a wide range of genes. In addition, they further suggest that defects in proliferation may not be caused by defective transcription.
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TFIIH is involved in a DNA repair mechanism known as NER (Hoeijmakers 2001), and loss of function of yeast and human p52 is associated with defective NER (Feaver et al. 1997; Marinoni et al. 1997). NER-deficient cells are generally hypersensitive to UV light and UV irradiation triggers apoptosis. In order to test whether mrn is required for the NER activity, we examined the UV sensitivity of mrn mutant cells in the eye imaginal disc. When larvae are irradiated with 500 J/m2 of UV light and dissected 4 h later, massive apoptosis is detected in mrn mutant clones (Fig. 4E,F), but not in normal cells (Fig. 4D). The degree of apoptosis increases in proportion to the UV dose. Thus, cells with reduced p52 subunit are hypersensitive to the UV light in Drosophila, as in other organisms. Despite this severe phenotype upon UV irradiation, only a small number of TUNEL positive cells are observed in mrn mutant clones in the absence of irradiation (Fig. 4B,C). Furthermore, mutations in other NER components show almost normal development and DNA synthesis (Boyd et al. 1982; Friedberg & Meira 2000; Calleja et al. 2001), implying that under normal environmental conditions a defect in DNA repair function does not necessarily lead to a cell growth phenotype. It is thus likely that the proliferation defect of mrn clones is not due to a failure to repair spontaneous DNA damage.
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Another possible cause for the proliferation defect of mrn mutant clones is that p52/mrn mutants may have reduced CAK activity, which regulates the cell cycle by phosphorylating CDKs. To address this possibility, we assayed cell cycle progression in mrn mutant cells. In the first instar larval CNS and eye imaginal disc, BrdU incorporation of mrn–/– individuals is reduced compared to normal animals of the same stage, but not entirely lost (Fig. 5B,D). The lack of a severe phenotype in the first instar stage may be due to the presence of a maternal supply of p52/MRN, and/or the relative insensitivity of cell division at this stage to the reduction in MRN levels.
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To investigate the consequence of a lack of p52/MRN in this process, we examined the expression of several cell cycle markers in the mrn mutant mosaic eye: Cyclin E (CycE), which triggers the G1/S transition, BrdU, which is incorporated at S-phase, Cyclin B (CycB), which is detected in cells that have passed the G2/M transition but have not yet divided, and phospho-H3, which is detected during mitosis. In normal eye imaginal discs these cell cycle markers show a strictly coordinated pattern in the second mitotic wave. CycE is detected just behind the morphogenetic furrow (Fig. 6A), BrdU incorporation begins at column 1 and continues to column 3 (Fig. 6D), CycB is detected from column 3 onwards with a progressive reduction in labeled cells in posterior areas (Fig. 6G), and phospho-H3 is detected in column 4 in cells undergoing mitosis in the second mitotic wave (Fig. 6J).
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Compared to a rather mild phenotype in the anterior region of the eye imaginal disc, posterior to the morphogenetic furrow, cell cycle progression is completely blocked at the G1/S transition; CycE accumulates to abnormally high levels, BrdU incorporation is absent, and CycB and phospho-H3 expression is decreased (Fig. 6). To demonstrate the differential effect of the mrn mutation on the first and the second mitotic wave, we counted the number of M-phase cells anterior and posterior to the morphogenetic furrow. This quantitative analysis shows that the posterior region, where the second mitotic wave takes place, is more sensitive to the reduction in MRN activity compared to the anterior region (Fig. 6P). This difference in sensitivity may be due to the difference in the nature of the cell division: only the second mitotic wave is accompanied by developmentally-regulated cell cycle synchronization. Thus, p52/MRN may be necessary for G1/S transitions that occur under specific regulatory control, possibly responding to specific developmental signaling pathways.
CDK7 functions in both G1/S and G2/M transitions
While the CAK subcomplex of TFIIH is known to be essential for cell cycle progression, p52 is not a component of this subcomplex but instead is a component of the TFIIH core complex (Rossignol et al. 1997; Yankulov & Bentley 1997; Tirode et al. 1999; Araújo et al. 2000; Vermeulen et al. 2000; Fisher 2005). Since p52 mutations affect the cell cycle, we reasoned that the absence of p52 may alter the integrity of holo-TFIIH, affecting CAK enzymatic activity. To determine whether our mrn mutants are similar to CAK mutants, we compared loss-of-function phenotypes of mrn to those of cdk7, the gene encoding the catalytic subunit of the CAK subcomplex.
To examine the consequence of reduced CAK activity in the eye imaginal disc, we used a temperature sensitive allele of cdk7 (Larochelle et al. 1998). The cdk7ts allele encodes a protein that is destabilized at the restrictive temperature. When cdk7ts animals are shifted to 29°, the size of the eye imaginal disc becomes smaller in proportion to the time that the animals are kept at this temperature. When maintained at 29 °C for 3 days, abnormalities are seen not only at M-phase but also at S-phase; both phospho-H3 staining and incorporation of BrdU are reduced (Fig. 7E,K). CycE staining is increased, accompanied by a reduction in CycB (Fig. 7B,H). When animals are returned to 22° for 24 h after a 3-day shift to 29°, cells in G2 remain arrested (CycB accumulation and small number of phospho-H3 cells), while G1 arrested cells are able to enter S-phase (Fig. 7C,F,I,L). Thus, a CAK functions at the G1/S transition in Drosophila, suggesting that the cell cycle defects seen in mrn mutants may be caused by defective CAK activity.
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If p52/MRN is required for CAK activity, other subunits of the TFIIH core may also be required for in vivo CAK activity and/or cell cycle regulation. In order to determine whether a helicase/ATPase subunit participates in cell cycle regulation, we assayed for cell cycle progression in mutant clones of TFIIH XPB, encoded by the haywire (hay) gene (Mounkes et al. 1992). We used two nonsense mutations of hay: hayrv3 and hayrv7, both of which have reduced levels of hay mRNA (Mounkes & Fuller 1999). Since these alleles are homozygous lethal, hay mutant clones were generated in eye imaginal discs using somatic recombination. For both alleles, only small clones were obtained in the third instar larval eye disc (Fig. 8A–C), suggesting that hay, like mrn, is essential for cell proliferation. As in mrn mutant clones, CycE accumulated to high levels in hay mutant clones (Fig. 8E,F), consistent with the idea that XPB is also required for cell cycle regulation. Similarities in phenotypes among cdk7, mrn and hay suggest that in vivo, an intact TFIIH core complex is required for cell cycle regulation.
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Discussion |
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TFIIH regulates the cell cycle as a holocomplex
The most striking phenotype observed in mrn mutant clones is a defect in cell cycle progression. Two distinct forms of cell divisions take place in normal eye imaginal disc; one occurs anterior to the morphogenetic furrow in which cell divides randomly, and the other occurs posterior to the morphogenetic furrow (second mitotic wave), where the cell cycle is coordinated with morphogenesis and ommatidial differentiation. In the mrn mutant clone, cell cycle progression anterior to the morphogenetic furrow is slower than in wild-type, while at the second mitotic wave, mrn mutant cells are incapable of synchronizing their cell cycles and performing coordinated patterns of cell cycle events. Since similar phenotypes are observed in a cdk7 mutant, the cell cycle defect observed in mrn mutant clones is likely to be a consequence of compromised activity of CDK7, the enzymatic component of CAK.
How does the p52 subunit of the TFIIH core affect CAK activity? Because the three subunits of CAK are sufficient to activate Cdks in vitro, it has been thought that cell cycle regulation is performed by the trimeric CAK subcomplex, independent of the TFIIH core (Fisher 2005). However, events such as E2F degradation, nuclear receptor activation and phosphorylation of the RNA polymerase CTD require an intact TFIIH holoenzyme (Roy et al. 1994; Makela et al. 1995; Serizawa et al. 1995; Shiekhattar et al. 1995; Vandel & Kouzarides 1999; Bastien et al. 2000; Chen et al. 2000; Keriel et al. 2002). Likewise, cell cycle regulation in vivo may also be mediated by the TFIIH holocomplex. In fact, the substrate specificity of CAK is altered by the association between Mat1 and the TFIIH core, and Cdk4 is phosphorylated by the TFIIH complex rather than the CAK subcomplex (Rossignol et al. 1997; Yankulov & Bentley 1997; Watanabe et al. 2000). Our results demonstrate that two of the TFIIH core subunits, p52 and XPB, are required for CDK7-dependent cell cycle regulation strongly suggests that in vivo, CAK activity is carried out by the TFIIH holocomplex. This may allow integration of various developmental signals, such as those present at the morphogenetic furrow, to control TFIIH activity and thus cell cycle progression and synchronization.
p52/MRN is necessary for the CAK activity in G1/S transition
Our work on p52/MRN has also revealed a new role of CAK in cell cycle regulation. Two classes of CAKs have so far been identified: monomeric Cak1p from budding yeast and the cdk7/cyclin H/MAT1 subcomplex of TFIIH from metazoans. While yeast CAK is important for both G1/S and G2/M transitions, in other metazoans including Drosophila, only a role in the G2/M transition had been identified (Kaldis et al. 1996; Thuret et al. 1996; Sutton & Freiman 1997; Larochelle et al. 1998; Saiz & Fisher 2002). Because both p52/mrn and cdk7 mutations cause a block in the G1/S transition (G1 arrest) at the morphogenetic furrow, our results demonstrate that CAK activity of TFIIH is also required for the G1/S transition during a developmentally-regulated cell cycle progression. A recent report also showed the requirement of CAK in the G1/S transition in a human cell line (Larochelle et al. 2007). The dual functions of CAK in both G1/S and G2/M transitions may be a conserved feature of TFIIH-mediated cell cycle regulation in metazoans.
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Experimental procedures |
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We used the following fly strains: mrn mutant lines mrn1, mrn2, mrn3, mrn4, mrn5, mrn6, were gifts from Margaret T. Fuller and are listed in Flybase <http://flybase.bio.indiana.edu/>. Df (3 L) BK10, which deletes the mrn locus, is described in Leicht & Bonner (1988). hayrv3 and hayrv7 (Mounkes & Fuller 1999) were obtained from Margaret T. Fuller. mrn mutant clones in the eye were made in the following genotype: y w ey-Flp: mrn FRT80/P[ub-GFP] P[w+]70C M (3 L) FRT80. A line containing the P[y+]70C FRT80 chromosome was used as a mrn+ control. The strain that carries a temperature sensitive allele of cdk7 (Df(1)JB254 P[w+ snf+, dhd+];+; P[w+ cdk7P140S]) is described in Larochelle et al. (1998). GBS-NZ (West 1998; Niwa et al. 2004) carries a NLS-lacZ fusion gene in the pGMR vector (Hay et al. 1994), and expresses nuclear ß-galactosidase in all cells posterior to the morphogenetic furrow. ey-GAL4 (Halder et al. 1998) was used for rescue experiments.
Molecular biology
The full-length p52 cDNA was cloned into the pUAS vector for transgenic rescue and over-expression experiments. The P-element in line ep(3) 0572 was excised by crossing to a genomic source of transposase activity, and chromosomes that lost the white+ marker were screened by PCR. Lines that exhibited amplification fragments smaller than normal size were chosen as potential mrn deletion mutants, and their deletion end points were determined by sequencing. The mutation sites of all mrn alleles were determined by sequencing a 2.7 Kbp genomic PCR fragment including the mrn coding region.
Histology
In situ hybridization and antibody staining were performed as described (Tomlinson & Ready 1987; Tautz & Pfeifle 1989; O’Neill & Bier 1994) with minor modifications. Antibodies used in this study were rabbit anti-GFP (CLONTECH, Mountain View, CA; used at a 1 : 500 dilution), mouse anti-GFP (CLONTECH, used at a 1 : 500 dilution), mouse anti-ß-galactosidase (CPL, West Chester, PA; used at a 1 : 1000 dilution), mouse anti-CycE 8B10 (used at a 1 : 3 dilution), mouse anti-CycB F2F4 (used at a 1 : 5 dilution), rabbit anti-phospho-H3 (Upstate Biotechnology, Lake Placid, NY; used at a 1 : 200 dilution), rat anti-ELAV MAb 7E8A10 (Developmental Studies Hybridoma Bank, developed by G.M. Rubin, used at a 1 : 4 dilution), mouse anti-PROS (Kauffmann et al. 1996, used at a 1 : 4 dilution), rabbit anti-ATO (Jarman et al. 1995, used at a 1 : 5000 dilution) and rabbit anti-BarH1 (Higashijima et al. 1992, used at a 1 : 30 dilution). TOTO-3 (Molecular Probes, Eugene, OR) was used at 0.5 µM. Mutant clone area was calculated by VH ANALYZER software (Keyence, Osaka, Japan).
BrdU incorporation
Tissue was dissected in S2 medium (SIGMA, St. Louis, MO) and incubated in 80 µg/mL BrdU in S2 medium at RT for 2 h. The tissue was then fixed with 4% PFA for double staining and Carnoy's fixative for single staining. BrdU was detected by mouse anti-BrdU (Zymed, San Francisco, CA; used at a 1 : 2 dilution), with signal amplification using the TSA Biotin system (Perkin Elmer Life Science, Waltham, MA). All secondary antibodies (FITC or Cy3, HRP conjugated) were used at 1 : 400 dilutions.
TUNEL
Apoptotic signals were detected by TACS 2TdT DAB in situ Apoptosis Detection Kit (TREVIGEN, Gaithersburg, MD).
UV sensitivity
UV-C irradiation was performed for third instar larva using a XL-1500 UV cross LINKER (SPECTRONICS Corporation, Westbury, NY). After irradiation, larvae were transferred to yeast food vials and incubated at 25 °C for 4 h before fixation.
Figure preparation
Figures were prepared according to "Barrier-free presentation that is friendly to colorblind people" <http://jfly.iam.u-tokyo.ac.jp/color/>.
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Acknowledgements |
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Footnotes |
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aPresent address: Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183-8526, Japan.
bPresent address: Division for Animal Research Resources, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan.
cPresent address: Department of Anatomy, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
* Correspondence: E-mail: yhiromi{at}lab.nig.ac.jp |
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Received: 6 July 2007
Accepted: 13 August 2007
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