- Email: [email protected]
The C. elegans Tousled-like Kinase (TLK-1) Has an Essential Role in Transcription
Current Biology, Vol. 13, 1921–1929, November 11, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2003.10.035
The C. elegans Tousled-like Kinase (TLK-1) Has an Essential Role in Transcription Zhenbo Han,1 Jennifer R. Saam,2 Henry P. Adams,1 Susan E. Mango,2 and Jill M. Schumacher1,3,* 1 Department of Molecular Genetics The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 2 Huntsman Cancer Institute University of Utah Salt Lake City, Utah 84112 3 Genes and Development Program Graduate School of Biomedical Sciences The University of Texas, Houston Houston, Texas 77030
Summary Background: The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair. Here, we used RNA mediated interference (RNAi) to determine the function of the C. elegans Tousled kinase (TLK-1) during embryonic development. Results: TLK-1-deficient embryos arrested with a phenotype reminiscent of embryos that are broadly defective in transcription, and the expression of several reporter genes was dramatically reduced in tlk-1(RNAi) embryos. Furthermore, posttranslational modifications of RNA polymerase II (RNAPII) and histone H3 that have been correlated with transcription elongation, phosphorylation of the RNAPII CTD at Serine 2, and methylation of histone H3 at Lysine 36 were found at significantly reduced levels in tlk-1(RNAi) embryos as compared to wild-type. Conclusions: These results reveal a surprising requirement for a Tousled-like kinase in transcriptional regulation during development, likely during the elongation phase. In addition, our results confirm that the link between RNAPII phosphorylation and histone H3 methylation previously observed in budding yeast is functionally conserved in metazoans. Introduction The phosphorylation of specific residues within the heptapeptide repeats found in the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) large subunit have been directly correlated with different stages of transcription [1, 2]. RNAPII phosphorylated at CTD Ser5 is found primarily at promoters and appears to act in transcription initiation while RNAPII phosphorylated at CTD Ser2 predominates once RNAPII has cleared the promoter for transcription elongation [1]. The differential phosphorylation of CTD Ser5 and Ser2 controls the binding of many of the factors required for transcription and mRNA processing [1, 3–5]. *Correspondence: [email protected]
Multiple cell cycle-regulated kinases have been implicated in CTD phosphorylation including CDK1, CDK7, CDK8, and CDK9 [2]. CDK7 and its partner cyclin, cyclin H, have been shown to have two conserved essential functions: regulation of transcription via phosphorylation of the RNAPII CTD at Ser5 and cell cycle progression via CAK (cyclin-dependent kinase activating kinase) activity [6–8]. CDK9 and cyclin T are components of a transcription factor, pTEFb, which phosphorylates the RNAPII CTD at Ser2 and promotes transcription elongation [9]. Experiments in C. elegans have revealed that CDK-7 and CDK-9 are essential for embryonic viability and are broadly required for embryonic transcription [8, 10]. Depletion of CDK-9 by RNAi-mediated interference (RNAi) resulted in an embryonic lethal phenotype that was grossly indistinguishable from that produced by RNAi of the large subunit of RNAP II (AMA-1 in C. elegans), namely developmental arrest at approximately the 100-cell stage in the absence of differentiation [10, 11]. Not surprisingly, RNAPII CTD phosphorylation at Ser2 and Ser5 was no longer found in ama-1(RNAi) embryos [10, 11]. Interestingly, while CTD Ser5 phosphorylation was not affected, phosphorylation of CTD Ser2 was not detectable in cdk-9(RNAi) embryos [10]. Here, we report that the C. elegans homolog of the Tousled kinases is required for appropriate transcription and CTD phosphorylation during C. elegans embryonic development. Mutations in the founding member of the Tousled family, Arabidopsis thaliana Tousled, result in numerous developmental defects that lead to a “tousled” appearance of various tissues [12]. Two human homologs (Tlk1 and Tlk2) were subsequently found to be cell cycle-regulated kinases that display maximum expression and activity during S phase [13]. A yeast two-hybrid screen and in vitro phosphorylation assays revealed that human Asf1a and Asf1b are in vitro substrates of the human Tlk kinases [14]. Along with Asf1, histone H3 was also found to be an in vitro substrate of human Tlk [15]. Although the molecular and cellular consequences of Asf1 and histone H3 phosphorylation by Tlk are unknown, Tlk activity was recently shown to be downregulated in response to S phase DNA damage via the ATM and Chk1 kinase pathways [16]. These results suggest that Tousled kinases may play a critical role in chromatin remodeling during DNA replication and repair. Our analysis of C. elegans Tousled (TLK-1) has revealed an unanticipated role for a Tousled kinase in transcription. Here, we report that C. elegans TLK-1 is required for appropriate transcription during C. elegans development and differentially affects the posttranslational modifications that have been correlated with transcription elongation, RNAPII CTD phosphorylation at Ser2, and methylation of histone H3 at Lysine 36. Results Cloning of a Tousled-like Kinase from C. elegans (TLK-1) Intrigued by a potential role for Tousled kinases in histone H3 Ser10 phosphorylation [15], we performed Blast
Current Biology 1922
searches of the C. elegans genome to identify a single locus, C07A9.3, predicted to encode a protein with significant homology to the Tousled family of kinases. A full-length C07A9.3 cDNA was PCR amplified from a C. elegans cDNA library using primers specific for SL1 (a trans-spliced leader RNA found at the 5⬘ end of the majority of C. elegans mRNAs) [17] and the predicted stop codon. DNA sequencing of this clone revealed that SL1 is spliced to nucleotide 37134 of the C07A9 cosmid sequence, resulting in an open reading frame (ORF) that starts at amino acid 272 of the C07A9.3 predicted protein sequence (GenBank). Thus, the true C07A9.3 ORF begins at predicted exon 6. In agreement with our data, no sequences corresponding to predicted exons 1–5 are present in the C. elegans EST database (http:// www.wormbase.org) and the most 5⬘ C07A9.3 EST sequence (yk143f9.5) also corresponds to exon 6. An alignment with the corrected C07A9.3 protein product and other Tousled family members is shown in Figure S1. Given the high degree of sequence conservation between C07A9.3 and Tousled kinases, we have named this protein TLK-1 (Tousled-like kinase). C. elegans TLK-1 differs from other family members in that there is an insertion of several polyglutamine domains near the N terminus of the protein (Figure S1, boxed), while the kinase domain is found in the highly conserved C terminus (Figure S1, underlined). To determine whether TLK-1 is an essential protein in C. elegans, RNA-mediated interference was performed by microinjection of double-stranded RNA (dsRNA) or feeding young adult hermaphrodites bacteria expressing dsRNA corresponding to the tlk-1 cDNA. tlk-1(RNAi) by either method resulted in fully penetrant embryonic lethality in the broods of treated animals. As similar results were obtained by injection or feeding, feeding results are shown except as noted below. Affected embryos no longer expressed detectable levels of the TLK-1 protein (Figures 1A and 1B) and arrested development with approximately 100 cells with no signs of differentiation (Figure 1B). As discussed below, this phenotype is highly reminiscent of the embryonic lethality seen when RNAPII-dependent transcription is disrupted.
Cell Cycle-Specific Expression and Localization of C. elegans TLK-1 To assess the expression and localization pattern of the TLK-1 protein during C. elegans embryogenesis, a rabbit polyclonal antibody was raised against a maltose binding protein-TLK-1 fusion protein (MBP-TLK-1). Western blot analysis of wild-type C. elegans embryo lysates probed with affinity-purified TLK-1 antisera revealed a single protein of the predicted size (108 kDa) (Figure 1A). Antibody recognition of this protein was entirely eliminated in lysates from tlk-1(RNAi) embryos (Figure 1A). Immunolocalization studies using affinity-purified TLK-1 antisera on fixed C. elegans embryos revealed that the TLK-1 protein was exclusively found in nuclei from the two-cell stage until morphogenesis (Figure 1B). Staining was completely eliminated in tlk-1(RNAi) embryos (Figure 1B) and entirely competed by preincubation of TLK-1 antisera with MBP-TLK-1 (data not shown). Examination of TLK-1 immunostaining with respect to the cell cycle
revealed that TLK-1 was highly expressed in interphase and prophase nuclei (Figure 1C). TLK-1 immunostaining diminished dramatically upon nuclear envelope breakdown and did not appear to be above background levels in mitotic cells from metaphase through telophase (Figure 1C). However, at this time, we cannot distinguish whether this is due to diffusion of nuclear TLK-1 or due to degradation of the TLK-1 protein during mitosis. TLK-1 was also detectable in adult germline nuclei, suggesting that the TLK-1 protein is maternally expressed (data not shown). TLK-1 Is Not Required for Mitotic Histone H3 Phosphorylation Yeast Ipl1, C. elegans AIR-2, and other Aurora B kinases are required for mitotic phosphorylation of histone H3 at Ser10 [18–21]. It has recently been reported that human TLK-1 can also phosphorylate this residue [15]. To test whether C. elegans TLK-1 is a histone H3 kinase, a peptide corresponding to the N-terminal tail of histone H3 was incubated with bacterially expressed recombinant MBP-TLK-1 or GST-AIR-2 and [␥-32P-ATP] in kinase buffer. The H3 peptide was strongly phosphorylated in the presence of GST-AIR-2, but not MBP-TLK-1 (Figure 2A). To confirm that MBP-TLK-1 is an active kinase in vitro, MBP-TLK-1 was incubated with the myelin basic protein (MYBP) in assay conditions identical to those in the above experiments. MBP-TLK-1 underwent autophosphorylation and could phosphorylate MYBP in vitro (Figure 2B). Since phosphorylation of histone H3 normally takes place in the context of nucleosomes, kinase assays were performed with nucleosomes assembled in vitro [22, 23]. GST-AIR-2 readily phosphorylated histone H3 in this assay, where as MBP-TLK-1 did not (Figure 2C). To determine whether phosphorylation of histone H3(S10) is dependent on TLK-1 expression in vivo, wildtype, tlk-1(RNAi) and air-2(RNAi) embryos were fixed and stained with a polyclonal antibody specific for this modification (pH3). As a fixation control, all embryos were counterstained with a monoclonal ␣-tubulin antibody. While pH3 immunostaining was not detectable in air-2(RNAi) embryos as expected [18], mitotic chromosomes were strongly stained by this antibody in wildtype and tlk-1(RNAi) embryos (Figure 2D). Western blot analysis of wild-type and tlk-1(RNAi) embryo extracts confirmed that the level of H3(S10) phosphorylation did not change in the absence of the TLK-1 kinase (Figure 2E). As the feeding method of RNAi used in these experiments may not be as effective in reducing gene expression as microinjection [24], tlk-1(RNAi) embryos produced by microinjection were also examined for pH3 staining. As above, no differences were detected in the level of pH3 staining between wild-type and tlk-1(RNAi) embryos (Figure S2). TLK-1 Affects RNAPII and Histone Modifications that Are Correlated with Transcription Elongation While C. elegans embryonic development is initiated by maternally derived proteins and mRNAs, zygotic RNAPIIdependent mRNA transcription begins around the fourcell stage [11, 25]. When RNAPII activity is inhibited,
A Role for TLK-1 in Transcription 1923
Figure 1. TLK-1 Is a Nuclear Protein that Is Highly Expressed during Interphase and Prophase (A) Western blot of protein extracts from C. elegans wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) probed with affinitypurified TLK-1 antisera. Protein loading was confirmed by probing with an ␣-tubulin antibody. (B) Wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies to TLK-1 and ␣-tubulin. Bar: 10 m. (C) Wild-type and tlk-1(RNAi) one- and two-cell embryos (produced by feeding dsRNA) fixed and stained with DAPI and antibodies to TLK-1 and ␣-tubulin were staged with respect to the cell cycle as assessed by DAPI and tubulin staining. Bar: 10 m.
embryonic development is arrested at approximately the 100-cell stage in the absence of differentiation [26, 27]. As described above, tlk-1(RNAi) embryos also arrest with approximately 100 undifferentiated cells, suggesting that transcription may be broadly disrupted in the absence of the TLK-1 kinase. To test this notion, tlk-1(RNAi) experiments were performed in C. elegans strains harboring transgenes in which the regulatory regions of RNAPII-dependent embryonically expressed genes had been fused to a GFP reporter. When her-
maphrodites were fed HT115 bacteria transformed with the RNAi feeding vector L440 without an insert, embryonic GFP expression was readily apparent in the four strains tested (med-1::GFP [28], end-3::GFP [29], elt2::GFP [30], and myo-2::GFP;pes-10::GFP [M. Edgley, J. Liu, D. Riddle, and A. Fire, personal communication]). GFP expression was not detectable in RNAPII/ama1(RNAi) embryos and was greatly reduced in tlk-1(RNAi) embryos (Figure 3). Altogether, these results support the conclusion that tlk-1(RNAi) embryonic lethality is
Current Biology 1924
Figure 2. TLK-1 Does Not Phosphorylate H3(S10) In Vitro or Affect H3(S10) Phosphorylation In Vivo (A) Results of filter binding assays where a peptide corresponding to the N-terminal tail of histone H3 was incubated alone (H3), with MBPTLK-1 (H3⫹MBP-TLK-1), or with GST-AIR-2 (H3⫹GST-AIR-2) in kinase buffer supplemented with [␥-32P]ATP. 32P incorporation was measured by scintillation counting (y axis). Controls included MBP-TLK-1 and GST-AIR-2 without H3 peptide. Bar: Standard deviation from three independent experiments. (B) MBP-TLK-1 was incubated with the myelin basic protein (MYBP) in kinase buffer supplemented with [␥-32P]ATP. Kinase reactions were separated by SDS-PAGE, blotted to nitrocellulose, and subjected to phosphoimage analysis to measure 32P incorporation and stained with Ponceau S to confirm protein loading.
A Role for TLK-1 in Transcription 1925
Figure 3. Transcription Is Reduced in tlk-1(RNAi) Embryos Transgenic C. elegans strains harboring the indicated promoter:GFP reporter constructs were fed HT115 bacteria, Ht115 bacteria expressing tlk-1 dsRNA, or HT115 expressing ama-1 dsRNA. Embryos were isolated and analyzed by DIC and immunofluoresence (FL) microscopy to assess GFP expression. Bar: 10 m.
consistent with a broad defect in embryonic mRNA transcription. To determine the steps at which the TLK-1 kinase may impact the regulation of transcription, RNAPII activity in wild-type and tlk-1(RNAi) embryos was measured with respect to RNAPII CTD Ser2 and Ser5 phosphorylation [11, 27]. In C. elegans and Drosophila embryos, CTD phosphorylation patterns are strongly associated with transcriptional activity [11]. Wild-type, tlk-1(RNAi), and RNAPII/ama-1(RNAi) embryos were fixed and stained with monoclonal antibodies specific for the large subunit of RNAPII and phosphorylated CTD Ser2 and Ser5 [11] (Figure 4A). All embryos were counterstained with an AIR-1-specific antibody as a fixation control (data not shown) [31]. In wild-type C. elegans embryos, RNAPII-, pSer2-, and pSer5-specific immunostaining was present in all interphase and prophase somatic nuclei starting at the four-cell stage, while the RNAPII antibody also labeled germline nuclei (Figure 4A, [11]). Immunostaining with all three antibodies was reduced by approximately 80% when expression of the large subunit of RNAPII was inhibited by RNAi (ama-1(RNAi)) (Figures 4A and 4B, and data not shown). In tlk-1(RNAi) embryos, the intensity of RNAPII and pSer5 immunostaining was similar to wild-type, while pSer2-specific immunostaining was reduced by approximately 40% (Figures 4A and 4B). To confirm these results, wild-type, ama-1(RNAi), and tlk-1(RNAi) embryo extracts were subjected to Western blot analysis with the same antibodies. Again,
while RNAPII expression and pSer5 levels were unchanged by tlk-1(RNAi), pSer2 levels were reduced approximately 40% with respect to wild-type (Figure S3). To determine whether tlk-1(RNAi) by microinjection might result in a stronger phenotype with regard to CTD phosphorylation, similar experiments were performed on tlk-1(RNAi) embryos from microinjected hermaphrodites. Comparable results were obtained, levels of pSer5 did not change (data not shown), and pSer2 staining was reduced to a similar extent as in tlk-1(RNAi) embryos produced via the feeding method (Figure S4). Altogether, these results indicate that while TLK-1 is not required for expression of the large subunit of RNAPII or phosphorylation of CTD Ser5, it does affect phosphorylation of CTD Ser2, suggesting that TLK-1 may be involved in regulating RNAPII activity during transcription elongation. The methylation of specific lysines in the histone H3 tail has been shown to play a significant role in transcription silencing, activation, and elongation [32]. Methylation of Lys4 (MetLys4) has been correlated with transcription activation, while MetLys9 has been associated with transcriptionally repressed heterochromatin [33– 36]. Furthermore, it has recently been shown that the methyltransferase responsible for methylation of histone H3 Lys36 is primarily associated with the elongating form of RNAPII, suggesting that this modification may be involved in transcription elongation or its regulation [37–39]. Since TLK-1 appears to impact RNAPII activity
(C) MBP-TLK-1 and GST-AIR-2 were incubated with nucleosomes in kinase buffer supplemented with [␥-32P]ATP. Kinase reactions were separated by SDS-PAGE, blotted to nitrocellulose, and subjected to phosphoimage analysis to measure 32P incorporation and stained with Ponceau S (histones) or subjected to Western blot analysis with TLK-1 and AIR-2 specific antibodies to confirm protein loading. (D) Wild-type, tlk-1(RNAi), and air-2(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies specific for histone H3(S10) phosphorylation (pH3) and ␣-tubulin. Bar: 10 m. (E) A Western blot of protein extracts from wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) was probed with pH3 and ␣-tubulin antibodies.
Current Biology 1926
Figure 4. RNAPII CTD Ser2 Phosphorylation Is Reduced in the Absence of TLK-1 (A) Wild-type, tlk-1(RNAi), and ama-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI, and antibodies for the large subunit of RNAPII, RNAPII CTD pSer5, and RNAPII CTD pSer2. Bar: 10 m. (B) Immunostaining intensity of embryos treated as in (A) was quantified as described in Experimental Procedures. Bar: standard deviation for measurements of 30–40 embryos per antibody.
with respect to transcription elongation, we examined the state of Lys36 methylation (MetLys36) in tlk-1(RNAi) embryos. Wild-type and tlk-1(RNAi) embryos were fixed and stained with polyclonal antibodies specific for histone H3 MetLys4, MetLys9, and MetLys36. As a fixation control, all embryos were counterstained with an ␣-tubulin-specific monoclonal antibody. While the intensity of MetLys4 and MetLys9 immunostaining did not change, MetLys36 staining in tlk-1(RNAi) embryos was reduced to approximately 60% of wild-type levels (Figures 5A and 5B). Western blot analysis of wild-type and tlk-1(RNAi) protein extracts confirmed there was a significant decrease in methylation of histone H3 Lys36 in tlk-1(RNAi) embryos (Figure S5). Altogether, these results are consistent with a role for the TLK-1 kinase in the regulation of transcription elongation. Discussion Here, we report that the C. elegans Tousled-like kinase TLK-1 is broadly essential for appropriate transcription
during embryonic development. Furthermore, distinct posttranslational modifications that have been linked to transcription elongation, namely phosphorylation of the RNAPII CTD at Ser2 and methylation of histone H3 at Lys36, are specifically reduced in TLK-1-deficient embryos. The human Tousled homologs have previously been shown to be cell cycle-regulated kinases with high levels of expression and kinase activity linked to ongoing DNA replication [13]. BrDU incorporation assays revealed no obvious defects in DNA replication in tlk-1(RNAi) embryos produced by feeding (Z.H. and J.M.S., unpublished data). However, a significant fraction of 50–100 cell tlk-1(RNAi) embryos from mothers microinjected with tlk-1 dsRNA had cells with aberrant chromatin morphology, including DNA bridges and brighter than wildtype DAPI staining (J.R.S. and S.E.M., unpublished data). A complete analysis of these defects is in progress. These results suggest that although no residual TLK-1 protein was detected in tlk-1(RNAi) embryos produced by feeding, an amount of TLK-1 activity sufficient
A Role for TLK-1 in Transcription 1927
Figure 5. Loss of TLK-1 Expression Affects Methylation of Histone H3 at Lys36 (A) Wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies for histone H3 MetLys4, MetLys9, and MetLys36. Bar: 10 m. (B) Immunostaining intensity of embryos treated as in (A) was quantified as described in the Supplemental Experimental Procedures. Bars: standard deviation for measurements of 30–40 embryos per antibody.
for grossly normal DNA replication and chromosome morphology was retained. Since the transcription phenotypes described here were consistent regardless of the RNAi method used, this implies that these affects are not simply secondary consequences of severe defects in DNA replication or chromosome integrity. Altogether, our data is consistent with a model whereby TLK-1 has two distinct and separable functions during C. elegans development. One function directly affects RNAPII activity and a second function ensures accurate DNA replication and/or chromatin assembly. Alternatively, TLK-1 may be involved in producing a chromatin template that differentially affects transcription and chromosome morphology. These models are not necessarily mutually exclusive and are discussed in detail below. A Direct Role for TLK-1 in Transcription? Distinct stages of transcription and RNA processing are temporally and spatially linked and are associated with differential RNAPII CTD phosphorylation [1]. RNAPII phosphorylated at CTD Ser5 is found at promoter regions and is associated with RNA-capping enzymes [40], while Ser2 phosphorylated RNAPII predominates in coding regions [1]. The transition from promoter clearance to active elongation complexes has been dubbed a transcription “checkpoint” that ensures that RNA pro-
duction and processing events take place in the required order [41]. To date, few genes are known to affect CTD Ser2 phosphorylation without affecting Ser5 phosphorylation [41]. Our results suggest that TLK-1 may be acting coincident with this checkpoint either by directly phosphorylating the CTD or perhaps by regulating the activity of a CTD Ser2-specific enzyme, such as the CTD Ser2 kinase CDK-9 or the CTD Ser2 phosphatase Fcp1 [10, 42, 43]. As noted above, Tousled-like kinases have been suggested to have role in DNA replication [13]. Separable roles for TLK-1 in the regulation of CTD phosphorylation and cell cycle control nicely parallel the functions of the CTD kinase CDK-7 [6–8]. CDK-7 phosphorylates CTD Ser5 and activates cyclin-dependent kinase complexes [6–8]. Depletion of C. elegans CDK-7 activity by a temperature-sensitive (ts) cdk-7 mutation or (cdk-7)RNAi results in embryonic lethality at the 50-cell stage that is characterized by loss of CTD Ser2 and Ser5 phosphorylation and transcription defects [8]. A fully penetrant block in cell cycle progression is only revealed when cdk-7(RNAi) is performed in the cdk-7(ts) mutant background. These conditions presumably eliminate CDK-7 activity and result in embryonic arrest at the one-cell stage [8]. As with CDK-7, our results with TLK-1 suggest that transcription may be more sensitive to alterations in the overall level of TLK-1 kinase activity than cell cycle events that may also require TLK-1.
Current Biology 1928
A recent flurry of papers have reported that the methyltransferase responsible for methylation of histone H3 Lys36 in yeast, Set2, preferentially associates with the CTD of RNAPII phosphorylated at Ser2, suggesting that methylation of histone H3 Lys36 is involved in the regulation of transcription elongation in S. cerevisiae [37–39, 44]. A variety of genetic and biochemical studies have provided evidence to support this conclusion. We found that histone H3 Lys36 methylation is reduced in tlk-1 (RNAi) embryos to the same extent as CTD Ser2 phosphorylation. These results provide compelling evidence that the link between histone H3 Lys36 methylation and CTD Ser2 phosphorylation is conserved in higher eukaryotes and support the notion that the transcription defects seen in TLK-1-deficient embryos are likely to be due to events occurring after RNAPII has cleared the promoter and has entered the elongation phase of transcription. Tousled and Chromatin Assembly Tlks have been implicated in chromatin regulation due to their ability to phosphorylate histone H3 and the histone chaperone Asf1 in vitro [14, 15]. We found no evidence that TLK-1 affects histone H3 phosphorylation in vitro or in vivo (regardless of the RNAi method used). These results suggest that TLK-1 does not directly contribute to the overall level of mitotic histone H3 phosphorylation during C. elegans embryogenesis. However, the possibility that TLK-1 is responsible for H3 phosphorylation at specific loci or promoters during interphase or other stages of the cell cycle cannot be ruled out. Asf1 and its partner complex, CAF-1, are required for the deposition of histones H3 and H4 during DNA replication and repair [45–47]. Asf1 appears to participate in the assembly of transcriptionally silent chromatin in yeast [45] and is required for the cell cycle-dependent expression of histone genes and cell cycle progression [45, 48]. Although the functional consequence of Asf phosphorylation by Tousled is unknown, loss of Tousled activity during DNA replication may result in altered Asf activity, leading to defects in the chromatin template. The chromatin integrity defects seen in tlk-1(RNAi) embryos produced by microinjection (J.R.S. and S.E.M., unpublished data) support the idea that TLK-1 has an essential role in building functional chromatin. Even in the absence of gross morphological chromatin defects, loss of TLK-1 activity is not compatible with appropriate transcription. Efficient transcription elongation in vitro requires the disruption of nucleosomes by FACT (Facilitates Chromatin Transcription), a protein complex that colocalizes with elongating RNAPII in vivo [41, 49]. Perhaps Tousled-mediated phosphorylation permits Asf and CAF to assemble chromatin that can be efficiently disassembled by FACT during transcription. Multiple Asf, CAF, and FACT subunit homologs exist in C. elegans, and we are currently addressing the relationship between these proteins and TLK-1 during C. elegans embryogenesis. Supplemental Data Supplemental Data including experimental procedures and additional figures are available http://www.current-biology.com/cgi/ content/full/13/22/1921/DC1/.
Acknowledgments We thank Y. Kohara for C. elegans cDNA clones; M. Barton, K. Shumway, and S. Dent for reagents; J. McGhee, G. Seydoux, J. Rothman, and the C. elegans Genetics Center for strains; R. Haynes for media preparation; T. Heallen for technical assistance; S. Dent and M. Barton for helpful discussions; and S. Dent, M. Barton, and members of the Schumacher lab for critical reading of the manuscript. DNA sequencing was performed at the MDACC sequencing core (supported by NCI Grant CA-16672). This work was supported by an NIH grants awarded to J.M.S. (R01 GM62181-01) and J.R.S (K01 DK02966-01A1). Received: June 6, 2003 Revised: September 18, 2003 Accepted: September 18, 2003 Published: November 11, 2003 References 1. Komarnitsky, P., Cho, E.J., and Buratowski, S. (2000). Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 2452–2460. 2. Kobor, M.S., and Greenblatt, J. (2002). Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta 1577, 261–275. 3. McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Shuman, S., and Bentley, D.L. (1997). 5⬘-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 3306–3318. 4. Licatalosi, D.D., Geiger, G., Minet, M., Schroeder, S., Cilli, K., McNeil, J.B., and Bentley, D.L. (2002). Functional interaction of yeast pre-mRNA 3⬘ end processing factors with RNA polymerase II. Mol. Cell 9, 1101–1111. 5. Oelgeschlager, T. (2002). Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control. J. Cell. Physiol. 190, 160–169. 6. Nigg, E.A. (1996). Cyclin-dependent kinase 7: at the cross-roads of transcription, DNA repair and cell cycle control? Curr. Opin. Cell Biol. 8, 312–317. 7. 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. 8. 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. 9. Price, D.H. (2000). P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20, 2629– 2634. 10. Shim, E.Y., Walker, A.K., Shi, Y., and Blackwell, T.K. (2002). CDK9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. Genes Dev. 16, 2135– 2146. 11. 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. 12. Roe, J.L., Rivin, C.J., Sessions, R.A., Feldmann, K.A., and Zambryski, P.C. (1993). The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 75, 939–950. 13. Sillje, H.H., Takahashi, K., Tanaka, K., Van Houwe, G., and Nigg, E.A. (1999). Mammalian homologues of the plant Tousled gene code for cell-cycle-regulated kinases with maximal activities linked to ongoing DNA replication. EMBO J. 18, 5691–5702. 14. Sillje, H.H., and Nigg, E.A. (2001). Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases. Curr. Biol. 11, 1068–1073.
A Role for TLK-1 in Transcription 1929
15. Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and De Benedetti, A. (2001). A translationally regulated Tousled kinase phosphorylates histone H3 and confers radioresistance when overexpressed. Oncogene 20, 726–738. 16. Groth, A., Lukas, J., Nigg, E.A., Sillje, H.H., Wernstedt, C., Bartek, J., and Hansen, K. (2003). Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint. EMBO J. 22, 1676–1687. 17. Krause, M., and Hirsh, D. (1987). A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49, 753–761. 18. Hsu, J.Y., Sun, Z.W., Li, X., Reuben, M., Tatchell, K., Bishop, D.K., Grushcow, J.M., Brame, C.J., Caldwell, J.A., Hunt, D.F., et al. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291. 19. Giet, R., and Glover, D.M. (2001). Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152, 669–682. 20. Murnion, M.E., Adams, R.R., Callister, D.M., Allis, C.D., Earnshaw, W.C., and Swedlow, J.R. (2001). Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation. J. Biol. Chem. 276, 26656–26665. 21. Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D., and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell. Biol. 22, 874–885. 22. Hayes, J.J., and Lee, K.M. (1997). In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12, 2–9. 23. Luger, K., Rechsteiner, T.J., and Richmond, T.J. (1999). Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19. 24. Timmons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. 25. Newman-Smith, E.D., and Rothman, J.H. (1998). The maternalto-zygotic transition in embryonic patterning of Caenorhabditis elegans. Curr. Opin. Genet. Dev. 8, 472–480. 26. Powell-Coffman, J.A., Knight, J., and Wood, W.B. (1996). Onset of C. elegans gastrulation is blocked by inhibition of embryonic transcription with an RNA polymerase antisense RNA. Dev. Biol. 178, 472–483. 27. Bird, D.M., and Riddle, D.L. (1989). Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II. Mol. Cell. Biol. 9, 4119–4130. 28. Maduro, M.F., Meneghini, M.D., Bowerman, B., BroitmanMaduro, G., and Rothman, J.H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol. Cell 7, 475–485. 29. Maduro, M.F., Lin, R., and Rothman, J.H. (2002). Dynamics of a developmental switch: recursive intracellular and intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wntinhibited transcriptional repression. Dev. Biol. 248, 128–142. 30. Fukushige, T., Hendzel, M.J., Bazett-Jones, D.P., and McGhee, J.D. (1999). Direct visualization of the elt-2 gut-specific GATA factor binding to a target promoter inside the living Caenorhabditis elegans embryo. Proc. Natl. Acad. Sci. USA 96, 11883– 11888. 31. Schumacher, J.M., Ashcroft, N., Donovan, P.J., and Golden, A. (1998). A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos. Development 125, 4391–4402. 32. Lachner, M., and Jenuwein, T. (2002). The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 14, 286–298. 33. Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Schneider, R., Bouman, P., Liu, J.S., Kouzarides, T., and Schreiber, S.L. (2002). Methylation of histone H3 Lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. USA 99, 8695–8700. 34. Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bernstein, B.E., Emre, N.C., Schreiber, S.L., Mellor, J., and Kou-
35.
36. 37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
48.
49.
zarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature 419, 407–411. Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., and Grewal, S.I. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113. Schreiber, S.L., and Bernstein, B.E. (2002). Signaling network model of chromatin. Cell 111, 771–778. Li, B., Howe, L., Anderson, S., Yates, J.R., 3rd, and Workman, J.L. (2003). The Set2 Histone Methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 278, 8897–8903. Li, J., Moazed, D., and Gygi, S.P. (2002). Association of the histone methyltransferase Set2 with RNA polymerase II plays a role in transcription elongation. J. Biol. Chem. 277, 49383– 49388. Krogan, N.J., Kim, M., Tong, A., Golshani, A., Cagney, G., Canadien, V., Richards, D.P., Beattie, B.K., Emili, A., Boone, C., et al. (2003). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23, 4207–4218. Fong, N., and Bentley, D.L. (2001). Capping, splicing, and 3⬘ processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD. Genes Dev. 15, 1783–1795. Orphanides, G., and Reinberg, D. (2002). A unified theory of gene expression. Cell 108, 439–451. Marshall, N.F., Peng, J., Xie, Z., and Price, D.H. (1996). Control of RNA polymerase II elongation potential by a novel carboxylterminal domain kinase. J. Biol. Chem. 271, 27176–27183. Kobor, M.S., Archambault, J., Lester, W., Holstege, F.C., Gileadi, O., Jansma, D.B., Jennings, E.G., Kouyoumdjian, F., Davidson, A.R., Young, R.A., et al. (1999). An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. Mol. Cell 4, 55–62. Xiao, T., Hall, H., Kizer, K.O., Shibata, Y., Hall, M.C., Borchers, C.H., and Strahl, B.D. (2003). Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast. Genes Dev. 17, 654–663. Tyler, J.K., Adams, C.R., Chen, S.R., Kobayashi, R., Kamakaka, R.T., and Kadonaga, J.T. (1999). The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555–560. Tyler, J.K., Collins, K.A., Prasad-Sinha, J., Amiott, E., Bulger, M., Harte, P.J., Kobayashi, R., and Kadonaga, J.T. (2001). Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Mol. Cell. Biol. 21, 6574–6584. Mello, J.A., Sillje, H.H., Roche, D.M., Kirschner, D.B., Nigg, E.A., and Almouzni, G. (2002). Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3, 329–334. Sutton, A., Bucaria, J., Osley, M.A., and Sternglanz, R. (2001). Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription. Genetics 158, 587–596. Saunders, A., Werner, J., Andrulis, E.D., Nakayama, T., Hirose, S., Reinberg, D., and Lis, J.T. (2003). Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo. Science 301, 1094–1096.
Accession Numbers Coordinates for the C. elegans TLK-1 cDNA sequence have been deposited under accession number AY450852.
The C. elegans Tousled-like Kinase (TLK-1) Has an Essential Role in Transcription Zhenbo Han,1 Jennifer R. Saam,2 Henry P. Adams,1 Susan E. Mango,2 and Jill M. Schumacher1,3,* 1 Department of Molecular Genetics The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030 2 Huntsman Cancer Institute University of Utah Salt Lake City, Utah 84112 3 Genes and Development Program Graduate School of Biomedical Sciences The University of Texas, Houston Houston, Texas 77030
Summary Background: The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair. Here, we used RNA mediated interference (RNAi) to determine the function of the C. elegans Tousled kinase (TLK-1) during embryonic development. Results: TLK-1-deficient embryos arrested with a phenotype reminiscent of embryos that are broadly defective in transcription, and the expression of several reporter genes was dramatically reduced in tlk-1(RNAi) embryos. Furthermore, posttranslational modifications of RNA polymerase II (RNAPII) and histone H3 that have been correlated with transcription elongation, phosphorylation of the RNAPII CTD at Serine 2, and methylation of histone H3 at Lysine 36 were found at significantly reduced levels in tlk-1(RNAi) embryos as compared to wild-type. Conclusions: These results reveal a surprising requirement for a Tousled-like kinase in transcriptional regulation during development, likely during the elongation phase. In addition, our results confirm that the link between RNAPII phosphorylation and histone H3 methylation previously observed in budding yeast is functionally conserved in metazoans. Introduction The phosphorylation of specific residues within the heptapeptide repeats found in the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) large subunit have been directly correlated with different stages of transcription [1, 2]. RNAPII phosphorylated at CTD Ser5 is found primarily at promoters and appears to act in transcription initiation while RNAPII phosphorylated at CTD Ser2 predominates once RNAPII has cleared the promoter for transcription elongation [1]. The differential phosphorylation of CTD Ser5 and Ser2 controls the binding of many of the factors required for transcription and mRNA processing [1, 3–5]. *Correspondence: [email protected]
Multiple cell cycle-regulated kinases have been implicated in CTD phosphorylation including CDK1, CDK7, CDK8, and CDK9 [2]. CDK7 and its partner cyclin, cyclin H, have been shown to have two conserved essential functions: regulation of transcription via phosphorylation of the RNAPII CTD at Ser5 and cell cycle progression via CAK (cyclin-dependent kinase activating kinase) activity [6–8]. CDK9 and cyclin T are components of a transcription factor, pTEFb, which phosphorylates the RNAPII CTD at Ser2 and promotes transcription elongation [9]. Experiments in C. elegans have revealed that CDK-7 and CDK-9 are essential for embryonic viability and are broadly required for embryonic transcription [8, 10]. Depletion of CDK-9 by RNAi-mediated interference (RNAi) resulted in an embryonic lethal phenotype that was grossly indistinguishable from that produced by RNAi of the large subunit of RNAP II (AMA-1 in C. elegans), namely developmental arrest at approximately the 100-cell stage in the absence of differentiation [10, 11]. Not surprisingly, RNAPII CTD phosphorylation at Ser2 and Ser5 was no longer found in ama-1(RNAi) embryos [10, 11]. Interestingly, while CTD Ser5 phosphorylation was not affected, phosphorylation of CTD Ser2 was not detectable in cdk-9(RNAi) embryos [10]. Here, we report that the C. elegans homolog of the Tousled kinases is required for appropriate transcription and CTD phosphorylation during C. elegans embryonic development. Mutations in the founding member of the Tousled family, Arabidopsis thaliana Tousled, result in numerous developmental defects that lead to a “tousled” appearance of various tissues [12]. Two human homologs (Tlk1 and Tlk2) were subsequently found to be cell cycle-regulated kinases that display maximum expression and activity during S phase [13]. A yeast two-hybrid screen and in vitro phosphorylation assays revealed that human Asf1a and Asf1b are in vitro substrates of the human Tlk kinases [14]. Along with Asf1, histone H3 was also found to be an in vitro substrate of human Tlk [15]. Although the molecular and cellular consequences of Asf1 and histone H3 phosphorylation by Tlk are unknown, Tlk activity was recently shown to be downregulated in response to S phase DNA damage via the ATM and Chk1 kinase pathways [16]. These results suggest that Tousled kinases may play a critical role in chromatin remodeling during DNA replication and repair. Our analysis of C. elegans Tousled (TLK-1) has revealed an unanticipated role for a Tousled kinase in transcription. Here, we report that C. elegans TLK-1 is required for appropriate transcription during C. elegans development and differentially affects the posttranslational modifications that have been correlated with transcription elongation, RNAPII CTD phosphorylation at Ser2, and methylation of histone H3 at Lysine 36. Results Cloning of a Tousled-like Kinase from C. elegans (TLK-1) Intrigued by a potential role for Tousled kinases in histone H3 Ser10 phosphorylation [15], we performed Blast
Current Biology 1922
searches of the C. elegans genome to identify a single locus, C07A9.3, predicted to encode a protein with significant homology to the Tousled family of kinases. A full-length C07A9.3 cDNA was PCR amplified from a C. elegans cDNA library using primers specific for SL1 (a trans-spliced leader RNA found at the 5⬘ end of the majority of C. elegans mRNAs) [17] and the predicted stop codon. DNA sequencing of this clone revealed that SL1 is spliced to nucleotide 37134 of the C07A9 cosmid sequence, resulting in an open reading frame (ORF) that starts at amino acid 272 of the C07A9.3 predicted protein sequence (GenBank). Thus, the true C07A9.3 ORF begins at predicted exon 6. In agreement with our data, no sequences corresponding to predicted exons 1–5 are present in the C. elegans EST database (http:// www.wormbase.org) and the most 5⬘ C07A9.3 EST sequence (yk143f9.5) also corresponds to exon 6. An alignment with the corrected C07A9.3 protein product and other Tousled family members is shown in Figure S1. Given the high degree of sequence conservation between C07A9.3 and Tousled kinases, we have named this protein TLK-1 (Tousled-like kinase). C. elegans TLK-1 differs from other family members in that there is an insertion of several polyglutamine domains near the N terminus of the protein (Figure S1, boxed), while the kinase domain is found in the highly conserved C terminus (Figure S1, underlined). To determine whether TLK-1 is an essential protein in C. elegans, RNA-mediated interference was performed by microinjection of double-stranded RNA (dsRNA) or feeding young adult hermaphrodites bacteria expressing dsRNA corresponding to the tlk-1 cDNA. tlk-1(RNAi) by either method resulted in fully penetrant embryonic lethality in the broods of treated animals. As similar results were obtained by injection or feeding, feeding results are shown except as noted below. Affected embryos no longer expressed detectable levels of the TLK-1 protein (Figures 1A and 1B) and arrested development with approximately 100 cells with no signs of differentiation (Figure 1B). As discussed below, this phenotype is highly reminiscent of the embryonic lethality seen when RNAPII-dependent transcription is disrupted.
Cell Cycle-Specific Expression and Localization of C. elegans TLK-1 To assess the expression and localization pattern of the TLK-1 protein during C. elegans embryogenesis, a rabbit polyclonal antibody was raised against a maltose binding protein-TLK-1 fusion protein (MBP-TLK-1). Western blot analysis of wild-type C. elegans embryo lysates probed with affinity-purified TLK-1 antisera revealed a single protein of the predicted size (108 kDa) (Figure 1A). Antibody recognition of this protein was entirely eliminated in lysates from tlk-1(RNAi) embryos (Figure 1A). Immunolocalization studies using affinity-purified TLK-1 antisera on fixed C. elegans embryos revealed that the TLK-1 protein was exclusively found in nuclei from the two-cell stage until morphogenesis (Figure 1B). Staining was completely eliminated in tlk-1(RNAi) embryos (Figure 1B) and entirely competed by preincubation of TLK-1 antisera with MBP-TLK-1 (data not shown). Examination of TLK-1 immunostaining with respect to the cell cycle
revealed that TLK-1 was highly expressed in interphase and prophase nuclei (Figure 1C). TLK-1 immunostaining diminished dramatically upon nuclear envelope breakdown and did not appear to be above background levels in mitotic cells from metaphase through telophase (Figure 1C). However, at this time, we cannot distinguish whether this is due to diffusion of nuclear TLK-1 or due to degradation of the TLK-1 protein during mitosis. TLK-1 was also detectable in adult germline nuclei, suggesting that the TLK-1 protein is maternally expressed (data not shown). TLK-1 Is Not Required for Mitotic Histone H3 Phosphorylation Yeast Ipl1, C. elegans AIR-2, and other Aurora B kinases are required for mitotic phosphorylation of histone H3 at Ser10 [18–21]. It has recently been reported that human TLK-1 can also phosphorylate this residue [15]. To test whether C. elegans TLK-1 is a histone H3 kinase, a peptide corresponding to the N-terminal tail of histone H3 was incubated with bacterially expressed recombinant MBP-TLK-1 or GST-AIR-2 and [␥-32P-ATP] in kinase buffer. The H3 peptide was strongly phosphorylated in the presence of GST-AIR-2, but not MBP-TLK-1 (Figure 2A). To confirm that MBP-TLK-1 is an active kinase in vitro, MBP-TLK-1 was incubated with the myelin basic protein (MYBP) in assay conditions identical to those in the above experiments. MBP-TLK-1 underwent autophosphorylation and could phosphorylate MYBP in vitro (Figure 2B). Since phosphorylation of histone H3 normally takes place in the context of nucleosomes, kinase assays were performed with nucleosomes assembled in vitro [22, 23]. GST-AIR-2 readily phosphorylated histone H3 in this assay, where as MBP-TLK-1 did not (Figure 2C). To determine whether phosphorylation of histone H3(S10) is dependent on TLK-1 expression in vivo, wildtype, tlk-1(RNAi) and air-2(RNAi) embryos were fixed and stained with a polyclonal antibody specific for this modification (pH3). As a fixation control, all embryos were counterstained with a monoclonal ␣-tubulin antibody. While pH3 immunostaining was not detectable in air-2(RNAi) embryos as expected [18], mitotic chromosomes were strongly stained by this antibody in wildtype and tlk-1(RNAi) embryos (Figure 2D). Western blot analysis of wild-type and tlk-1(RNAi) embryo extracts confirmed that the level of H3(S10) phosphorylation did not change in the absence of the TLK-1 kinase (Figure 2E). As the feeding method of RNAi used in these experiments may not be as effective in reducing gene expression as microinjection [24], tlk-1(RNAi) embryos produced by microinjection were also examined for pH3 staining. As above, no differences were detected in the level of pH3 staining between wild-type and tlk-1(RNAi) embryos (Figure S2). TLK-1 Affects RNAPII and Histone Modifications that Are Correlated with Transcription Elongation While C. elegans embryonic development is initiated by maternally derived proteins and mRNAs, zygotic RNAPIIdependent mRNA transcription begins around the fourcell stage [11, 25]. When RNAPII activity is inhibited,
A Role for TLK-1 in Transcription 1923
Figure 1. TLK-1 Is a Nuclear Protein that Is Highly Expressed during Interphase and Prophase (A) Western blot of protein extracts from C. elegans wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) probed with affinitypurified TLK-1 antisera. Protein loading was confirmed by probing with an ␣-tubulin antibody. (B) Wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies to TLK-1 and ␣-tubulin. Bar: 10 m. (C) Wild-type and tlk-1(RNAi) one- and two-cell embryos (produced by feeding dsRNA) fixed and stained with DAPI and antibodies to TLK-1 and ␣-tubulin were staged with respect to the cell cycle as assessed by DAPI and tubulin staining. Bar: 10 m.
embryonic development is arrested at approximately the 100-cell stage in the absence of differentiation [26, 27]. As described above, tlk-1(RNAi) embryos also arrest with approximately 100 undifferentiated cells, suggesting that transcription may be broadly disrupted in the absence of the TLK-1 kinase. To test this notion, tlk-1(RNAi) experiments were performed in C. elegans strains harboring transgenes in which the regulatory regions of RNAPII-dependent embryonically expressed genes had been fused to a GFP reporter. When her-
maphrodites were fed HT115 bacteria transformed with the RNAi feeding vector L440 without an insert, embryonic GFP expression was readily apparent in the four strains tested (med-1::GFP [28], end-3::GFP [29], elt2::GFP [30], and myo-2::GFP;pes-10::GFP [M. Edgley, J. Liu, D. Riddle, and A. Fire, personal communication]). GFP expression was not detectable in RNAPII/ama1(RNAi) embryos and was greatly reduced in tlk-1(RNAi) embryos (Figure 3). Altogether, these results support the conclusion that tlk-1(RNAi) embryonic lethality is
Current Biology 1924
Figure 2. TLK-1 Does Not Phosphorylate H3(S10) In Vitro or Affect H3(S10) Phosphorylation In Vivo (A) Results of filter binding assays where a peptide corresponding to the N-terminal tail of histone H3 was incubated alone (H3), with MBPTLK-1 (H3⫹MBP-TLK-1), or with GST-AIR-2 (H3⫹GST-AIR-2) in kinase buffer supplemented with [␥-32P]ATP. 32P incorporation was measured by scintillation counting (y axis). Controls included MBP-TLK-1 and GST-AIR-2 without H3 peptide. Bar: Standard deviation from three independent experiments. (B) MBP-TLK-1 was incubated with the myelin basic protein (MYBP) in kinase buffer supplemented with [␥-32P]ATP. Kinase reactions were separated by SDS-PAGE, blotted to nitrocellulose, and subjected to phosphoimage analysis to measure 32P incorporation and stained with Ponceau S to confirm protein loading.
A Role for TLK-1 in Transcription 1925
Figure 3. Transcription Is Reduced in tlk-1(RNAi) Embryos Transgenic C. elegans strains harboring the indicated promoter:GFP reporter constructs were fed HT115 bacteria, Ht115 bacteria expressing tlk-1 dsRNA, or HT115 expressing ama-1 dsRNA. Embryos were isolated and analyzed by DIC and immunofluoresence (FL) microscopy to assess GFP expression. Bar: 10 m.
consistent with a broad defect in embryonic mRNA transcription. To determine the steps at which the TLK-1 kinase may impact the regulation of transcription, RNAPII activity in wild-type and tlk-1(RNAi) embryos was measured with respect to RNAPII CTD Ser2 and Ser5 phosphorylation [11, 27]. In C. elegans and Drosophila embryos, CTD phosphorylation patterns are strongly associated with transcriptional activity [11]. Wild-type, tlk-1(RNAi), and RNAPII/ama-1(RNAi) embryos were fixed and stained with monoclonal antibodies specific for the large subunit of RNAPII and phosphorylated CTD Ser2 and Ser5 [11] (Figure 4A). All embryos were counterstained with an AIR-1-specific antibody as a fixation control (data not shown) [31]. In wild-type C. elegans embryos, RNAPII-, pSer2-, and pSer5-specific immunostaining was present in all interphase and prophase somatic nuclei starting at the four-cell stage, while the RNAPII antibody also labeled germline nuclei (Figure 4A, [11]). Immunostaining with all three antibodies was reduced by approximately 80% when expression of the large subunit of RNAPII was inhibited by RNAi (ama-1(RNAi)) (Figures 4A and 4B, and data not shown). In tlk-1(RNAi) embryos, the intensity of RNAPII and pSer5 immunostaining was similar to wild-type, while pSer2-specific immunostaining was reduced by approximately 40% (Figures 4A and 4B). To confirm these results, wild-type, ama-1(RNAi), and tlk-1(RNAi) embryo extracts were subjected to Western blot analysis with the same antibodies. Again,
while RNAPII expression and pSer5 levels were unchanged by tlk-1(RNAi), pSer2 levels were reduced approximately 40% with respect to wild-type (Figure S3). To determine whether tlk-1(RNAi) by microinjection might result in a stronger phenotype with regard to CTD phosphorylation, similar experiments were performed on tlk-1(RNAi) embryos from microinjected hermaphrodites. Comparable results were obtained, levels of pSer5 did not change (data not shown), and pSer2 staining was reduced to a similar extent as in tlk-1(RNAi) embryos produced via the feeding method (Figure S4). Altogether, these results indicate that while TLK-1 is not required for expression of the large subunit of RNAPII or phosphorylation of CTD Ser5, it does affect phosphorylation of CTD Ser2, suggesting that TLK-1 may be involved in regulating RNAPII activity during transcription elongation. The methylation of specific lysines in the histone H3 tail has been shown to play a significant role in transcription silencing, activation, and elongation [32]. Methylation of Lys4 (MetLys4) has been correlated with transcription activation, while MetLys9 has been associated with transcriptionally repressed heterochromatin [33– 36]. Furthermore, it has recently been shown that the methyltransferase responsible for methylation of histone H3 Lys36 is primarily associated with the elongating form of RNAPII, suggesting that this modification may be involved in transcription elongation or its regulation [37–39]. Since TLK-1 appears to impact RNAPII activity
(C) MBP-TLK-1 and GST-AIR-2 were incubated with nucleosomes in kinase buffer supplemented with [␥-32P]ATP. Kinase reactions were separated by SDS-PAGE, blotted to nitrocellulose, and subjected to phosphoimage analysis to measure 32P incorporation and stained with Ponceau S (histones) or subjected to Western blot analysis with TLK-1 and AIR-2 specific antibodies to confirm protein loading. (D) Wild-type, tlk-1(RNAi), and air-2(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies specific for histone H3(S10) phosphorylation (pH3) and ␣-tubulin. Bar: 10 m. (E) A Western blot of protein extracts from wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) was probed with pH3 and ␣-tubulin antibodies.
Current Biology 1926
Figure 4. RNAPII CTD Ser2 Phosphorylation Is Reduced in the Absence of TLK-1 (A) Wild-type, tlk-1(RNAi), and ama-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI, and antibodies for the large subunit of RNAPII, RNAPII CTD pSer5, and RNAPII CTD pSer2. Bar: 10 m. (B) Immunostaining intensity of embryos treated as in (A) was quantified as described in Experimental Procedures. Bar: standard deviation for measurements of 30–40 embryos per antibody.
with respect to transcription elongation, we examined the state of Lys36 methylation (MetLys36) in tlk-1(RNAi) embryos. Wild-type and tlk-1(RNAi) embryos were fixed and stained with polyclonal antibodies specific for histone H3 MetLys4, MetLys9, and MetLys36. As a fixation control, all embryos were counterstained with an ␣-tubulin-specific monoclonal antibody. While the intensity of MetLys4 and MetLys9 immunostaining did not change, MetLys36 staining in tlk-1(RNAi) embryos was reduced to approximately 60% of wild-type levels (Figures 5A and 5B). Western blot analysis of wild-type and tlk-1(RNAi) protein extracts confirmed there was a significant decrease in methylation of histone H3 Lys36 in tlk-1(RNAi) embryos (Figure S5). Altogether, these results are consistent with a role for the TLK-1 kinase in the regulation of transcription elongation. Discussion Here, we report that the C. elegans Tousled-like kinase TLK-1 is broadly essential for appropriate transcription
during embryonic development. Furthermore, distinct posttranslational modifications that have been linked to transcription elongation, namely phosphorylation of the RNAPII CTD at Ser2 and methylation of histone H3 at Lys36, are specifically reduced in TLK-1-deficient embryos. The human Tousled homologs have previously been shown to be cell cycle-regulated kinases with high levels of expression and kinase activity linked to ongoing DNA replication [13]. BrDU incorporation assays revealed no obvious defects in DNA replication in tlk-1(RNAi) embryos produced by feeding (Z.H. and J.M.S., unpublished data). However, a significant fraction of 50–100 cell tlk-1(RNAi) embryos from mothers microinjected with tlk-1 dsRNA had cells with aberrant chromatin morphology, including DNA bridges and brighter than wildtype DAPI staining (J.R.S. and S.E.M., unpublished data). A complete analysis of these defects is in progress. These results suggest that although no residual TLK-1 protein was detected in tlk-1(RNAi) embryos produced by feeding, an amount of TLK-1 activity sufficient
A Role for TLK-1 in Transcription 1927
Figure 5. Loss of TLK-1 Expression Affects Methylation of Histone H3 at Lys36 (A) Wild-type and tlk-1(RNAi) embryos (produced by feeding dsRNA) were fixed and stained with DAPI and antibodies for histone H3 MetLys4, MetLys9, and MetLys36. Bar: 10 m. (B) Immunostaining intensity of embryos treated as in (A) was quantified as described in the Supplemental Experimental Procedures. Bars: standard deviation for measurements of 30–40 embryos per antibody.
for grossly normal DNA replication and chromosome morphology was retained. Since the transcription phenotypes described here were consistent regardless of the RNAi method used, this implies that these affects are not simply secondary consequences of severe defects in DNA replication or chromosome integrity. Altogether, our data is consistent with a model whereby TLK-1 has two distinct and separable functions during C. elegans development. One function directly affects RNAPII activity and a second function ensures accurate DNA replication and/or chromatin assembly. Alternatively, TLK-1 may be involved in producing a chromatin template that differentially affects transcription and chromosome morphology. These models are not necessarily mutually exclusive and are discussed in detail below. A Direct Role for TLK-1 in Transcription? Distinct stages of transcription and RNA processing are temporally and spatially linked and are associated with differential RNAPII CTD phosphorylation [1]. RNAPII phosphorylated at CTD Ser5 is found at promoter regions and is associated with RNA-capping enzymes [40], while Ser2 phosphorylated RNAPII predominates in coding regions [1]. The transition from promoter clearance to active elongation complexes has been dubbed a transcription “checkpoint” that ensures that RNA pro-
duction and processing events take place in the required order [41]. To date, few genes are known to affect CTD Ser2 phosphorylation without affecting Ser5 phosphorylation [41]. Our results suggest that TLK-1 may be acting coincident with this checkpoint either by directly phosphorylating the CTD or perhaps by regulating the activity of a CTD Ser2-specific enzyme, such as the CTD Ser2 kinase CDK-9 or the CTD Ser2 phosphatase Fcp1 [10, 42, 43]. As noted above, Tousled-like kinases have been suggested to have role in DNA replication [13]. Separable roles for TLK-1 in the regulation of CTD phosphorylation and cell cycle control nicely parallel the functions of the CTD kinase CDK-7 [6–8]. CDK-7 phosphorylates CTD Ser5 and activates cyclin-dependent kinase complexes [6–8]. Depletion of C. elegans CDK-7 activity by a temperature-sensitive (ts) cdk-7 mutation or (cdk-7)RNAi results in embryonic lethality at the 50-cell stage that is characterized by loss of CTD Ser2 and Ser5 phosphorylation and transcription defects [8]. A fully penetrant block in cell cycle progression is only revealed when cdk-7(RNAi) is performed in the cdk-7(ts) mutant background. These conditions presumably eliminate CDK-7 activity and result in embryonic arrest at the one-cell stage [8]. As with CDK-7, our results with TLK-1 suggest that transcription may be more sensitive to alterations in the overall level of TLK-1 kinase activity than cell cycle events that may also require TLK-1.
Current Biology 1928
A recent flurry of papers have reported that the methyltransferase responsible for methylation of histone H3 Lys36 in yeast, Set2, preferentially associates with the CTD of RNAPII phosphorylated at Ser2, suggesting that methylation of histone H3 Lys36 is involved in the regulation of transcription elongation in S. cerevisiae [37–39, 44]. A variety of genetic and biochemical studies have provided evidence to support this conclusion. We found that histone H3 Lys36 methylation is reduced in tlk-1 (RNAi) embryos to the same extent as CTD Ser2 phosphorylation. These results provide compelling evidence that the link between histone H3 Lys36 methylation and CTD Ser2 phosphorylation is conserved in higher eukaryotes and support the notion that the transcription defects seen in TLK-1-deficient embryos are likely to be due to events occurring after RNAPII has cleared the promoter and has entered the elongation phase of transcription. Tousled and Chromatin Assembly Tlks have been implicated in chromatin regulation due to their ability to phosphorylate histone H3 and the histone chaperone Asf1 in vitro [14, 15]. We found no evidence that TLK-1 affects histone H3 phosphorylation in vitro or in vivo (regardless of the RNAi method used). These results suggest that TLK-1 does not directly contribute to the overall level of mitotic histone H3 phosphorylation during C. elegans embryogenesis. However, the possibility that TLK-1 is responsible for H3 phosphorylation at specific loci or promoters during interphase or other stages of the cell cycle cannot be ruled out. Asf1 and its partner complex, CAF-1, are required for the deposition of histones H3 and H4 during DNA replication and repair [45–47]. Asf1 appears to participate in the assembly of transcriptionally silent chromatin in yeast [45] and is required for the cell cycle-dependent expression of histone genes and cell cycle progression [45, 48]. Although the functional consequence of Asf phosphorylation by Tousled is unknown, loss of Tousled activity during DNA replication may result in altered Asf activity, leading to defects in the chromatin template. The chromatin integrity defects seen in tlk-1(RNAi) embryos produced by microinjection (J.R.S. and S.E.M., unpublished data) support the idea that TLK-1 has an essential role in building functional chromatin. Even in the absence of gross morphological chromatin defects, loss of TLK-1 activity is not compatible with appropriate transcription. Efficient transcription elongation in vitro requires the disruption of nucleosomes by FACT (Facilitates Chromatin Transcription), a protein complex that colocalizes with elongating RNAPII in vivo [41, 49]. Perhaps Tousled-mediated phosphorylation permits Asf and CAF to assemble chromatin that can be efficiently disassembled by FACT during transcription. Multiple Asf, CAF, and FACT subunit homologs exist in C. elegans, and we are currently addressing the relationship between these proteins and TLK-1 during C. elegans embryogenesis. Supplemental Data Supplemental Data including experimental procedures and additional figures are available http://www.current-biology.com/cgi/ content/full/13/22/1921/DC1/.
Acknowledgments We thank Y. Kohara for C. elegans cDNA clones; M. Barton, K. Shumway, and S. Dent for reagents; J. McGhee, G. Seydoux, J. Rothman, and the C. elegans Genetics Center for strains; R. Haynes for media preparation; T. Heallen for technical assistance; S. Dent and M. Barton for helpful discussions; and S. Dent, M. Barton, and members of the Schumacher lab for critical reading of the manuscript. DNA sequencing was performed at the MDACC sequencing core (supported by NCI Grant CA-16672). This work was supported by an NIH grants awarded to J.M.S. (R01 GM62181-01) and J.R.S (K01 DK02966-01A1). Received: June 6, 2003 Revised: September 18, 2003 Accepted: September 18, 2003 Published: November 11, 2003 References 1. Komarnitsky, P., Cho, E.J., and Buratowski, S. (2000). Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 2452–2460. 2. Kobor, M.S., and Greenblatt, J. (2002). Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta 1577, 261–275. 3. McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Shuman, S., and Bentley, D.L. (1997). 5⬘-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 3306–3318. 4. Licatalosi, D.D., Geiger, G., Minet, M., Schroeder, S., Cilli, K., McNeil, J.B., and Bentley, D.L. (2002). Functional interaction of yeast pre-mRNA 3⬘ end processing factors with RNA polymerase II. Mol. Cell 9, 1101–1111. 5. Oelgeschlager, T. (2002). Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control. J. Cell. Physiol. 190, 160–169. 6. Nigg, E.A. (1996). Cyclin-dependent kinase 7: at the cross-roads of transcription, DNA repair and cell cycle control? Curr. Opin. Cell Biol. 8, 312–317. 7. 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. 8. 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. 9. Price, D.H. (2000). P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20, 2629– 2634. 10. Shim, E.Y., Walker, A.K., Shi, Y., and Blackwell, T.K. (2002). CDK9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. Genes Dev. 16, 2135– 2146. 11. 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. 12. Roe, J.L., Rivin, C.J., Sessions, R.A., Feldmann, K.A., and Zambryski, P.C. (1993). The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 75, 939–950. 13. Sillje, H.H., Takahashi, K., Tanaka, K., Van Houwe, G., and Nigg, E.A. (1999). Mammalian homologues of the plant Tousled gene code for cell-cycle-regulated kinases with maximal activities linked to ongoing DNA replication. EMBO J. 18, 5691–5702. 14. Sillje, H.H., and Nigg, E.A. (2001). Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases. Curr. Biol. 11, 1068–1073.
A Role for TLK-1 in Transcription 1929
15. Li, Y., DeFatta, R., Anthony, C., Sunavala, G., and De Benedetti, A. (2001). A translationally regulated Tousled kinase phosphorylates histone H3 and confers radioresistance when overexpressed. Oncogene 20, 726–738. 16. Groth, A., Lukas, J., Nigg, E.A., Sillje, H.H., Wernstedt, C., Bartek, J., and Hansen, K. (2003). Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint. EMBO J. 22, 1676–1687. 17. Krause, M., and Hirsh, D. (1987). A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49, 753–761. 18. Hsu, J.Y., Sun, Z.W., Li, X., Reuben, M., Tatchell, K., Bishop, D.K., Grushcow, J.M., Brame, C.J., Caldwell, J.A., Hunt, D.F., et al. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291. 19. Giet, R., and Glover, D.M. (2001). Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152, 669–682. 20. Murnion, M.E., Adams, R.R., Callister, D.M., Allis, C.D., Earnshaw, W.C., and Swedlow, J.R. (2001). Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation. J. Biol. Chem. 276, 26656–26665. 21. Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D., and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell. Biol. 22, 874–885. 22. Hayes, J.J., and Lee, K.M. (1997). In vitro reconstitution and analysis of mononucleosomes containing defined DNAs and proteins. Methods 12, 2–9. 23. Luger, K., Rechsteiner, T.J., and Richmond, T.J. (1999). Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19. 24. Timmons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. 25. Newman-Smith, E.D., and Rothman, J.H. (1998). The maternalto-zygotic transition in embryonic patterning of Caenorhabditis elegans. Curr. Opin. Genet. Dev. 8, 472–480. 26. Powell-Coffman, J.A., Knight, J., and Wood, W.B. (1996). Onset of C. elegans gastrulation is blocked by inhibition of embryonic transcription with an RNA polymerase antisense RNA. Dev. Biol. 178, 472–483. 27. Bird, D.M., and Riddle, D.L. (1989). Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II. Mol. Cell. Biol. 9, 4119–4130. 28. Maduro, M.F., Meneghini, M.D., Bowerman, B., BroitmanMaduro, G., and Rothman, J.H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol. Cell 7, 475–485. 29. Maduro, M.F., Lin, R., and Rothman, J.H. (2002). Dynamics of a developmental switch: recursive intracellular and intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wntinhibited transcriptional repression. Dev. Biol. 248, 128–142. 30. Fukushige, T., Hendzel, M.J., Bazett-Jones, D.P., and McGhee, J.D. (1999). Direct visualization of the elt-2 gut-specific GATA factor binding to a target promoter inside the living Caenorhabditis elegans embryo. Proc. Natl. Acad. Sci. USA 96, 11883– 11888. 31. Schumacher, J.M., Ashcroft, N., Donovan, P.J., and Golden, A. (1998). A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos. Development 125, 4391–4402. 32. Lachner, M., and Jenuwein, T. (2002). The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 14, 286–298. 33. Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Schneider, R., Bouman, P., Liu, J.S., Kouzarides, T., and Schreiber, S.L. (2002). Methylation of histone H3 Lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. USA 99, 8695–8700. 34. Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bernstein, B.E., Emre, N.C., Schreiber, S.L., Mellor, J., and Kou-
35.
36. 37.
38.
39.
40.
41. 42.
43.
44.
45.
46.
47.
48.
49.
zarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature 419, 407–411. Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., and Grewal, S.I. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113. Schreiber, S.L., and Bernstein, B.E. (2002). Signaling network model of chromatin. Cell 111, 771–778. Li, B., Howe, L., Anderson, S., Yates, J.R., 3rd, and Workman, J.L. (2003). The Set2 Histone Methyltransferase functions through the phosphorylated carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 278, 8897–8903. Li, J., Moazed, D., and Gygi, S.P. (2002). Association of the histone methyltransferase Set2 with RNA polymerase II plays a role in transcription elongation. J. Biol. Chem. 277, 49383– 49388. Krogan, N.J., Kim, M., Tong, A., Golshani, A., Cagney, G., Canadien, V., Richards, D.P., Beattie, B.K., Emili, A., Boone, C., et al. (2003). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23, 4207–4218. Fong, N., and Bentley, D.L. (2001). Capping, splicing, and 3⬘ processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD. Genes Dev. 15, 1783–1795. Orphanides, G., and Reinberg, D. (2002). A unified theory of gene expression. Cell 108, 439–451. Marshall, N.F., Peng, J., Xie, Z., and Price, D.H. (1996). Control of RNA polymerase II elongation potential by a novel carboxylterminal domain kinase. J. Biol. Chem. 271, 27176–27183. Kobor, M.S., Archambault, J., Lester, W., Holstege, F.C., Gileadi, O., Jansma, D.B., Jennings, E.G., Kouyoumdjian, F., Davidson, A.R., Young, R.A., et al. (1999). An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. Mol. Cell 4, 55–62. Xiao, T., Hall, H., Kizer, K.O., Shibata, Y., Hall, M.C., Borchers, C.H., and Strahl, B.D. (2003). Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast. Genes Dev. 17, 654–663. Tyler, J.K., Adams, C.R., Chen, S.R., Kobayashi, R., Kamakaka, R.T., and Kadonaga, J.T. (1999). The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555–560. Tyler, J.K., Collins, K.A., Prasad-Sinha, J., Amiott, E., Bulger, M., Harte, P.J., Kobayashi, R., and Kadonaga, J.T. (2001). Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Mol. Cell. Biol. 21, 6574–6584. Mello, J.A., Sillje, H.H., Roche, D.M., Kirschner, D.B., Nigg, E.A., and Almouzni, G. (2002). Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3, 329–334. Sutton, A., Bucaria, J., Osley, M.A., and Sternglanz, R. (2001). Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription. Genetics 158, 587–596. Saunders, A., Werner, J., Andrulis, E.D., Nakayama, T., Hirose, S., Reinberg, D., and Lis, J.T. (2003). Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo. Science 301, 1094–1096.
Accession Numbers Coordinates for the C. elegans TLK-1 cDNA sequence have been deposited under accession number AY450852.