FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1969606627> ?p ?o ?g. }

• W1969606627 endingPage "5850" @default.
• W1969606627 startingPage "5841" @default.
• W1969606627 abstract "Article3 November 2003free access TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo Jennifer A. Fairley Jennifer A. Fairley Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Pamela H. Scott Pamela H. Scott Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Robert J. White Corresponding Author Robert J. White Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Jennifer A. Fairley Jennifer A. Fairley Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Pamela H. Scott Pamela H. Scott Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Robert J. White Corresponding Author Robert J. White Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Author Information Jennifer A. Fairley1, Pamela H. Scott1 and Robert J. White 1 1Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5841-5850https://doi.org/10.1093/emboj/cdg544 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mitosis involves a generalized repression of gene expression. In the case of RNA polymerase III transcription, this is due to phosphorylation-mediated inactivation of TFIIIB, an essential complex comprising the TATA-binding protein TBP and the TAF subunits Brf1 and Bdp1. In HeLa cells, this repression is mediated by a mitotic kinase other than cdc2–cyclin B and is antagonized by protein phosphatase 2A. Brf1 is hyperphosphorylated in metaphase-arrested cells, but remains associated with promoters in condensed chromosomes, along with TBP. In contrast, Bdp1 is selectively released. Repression can be reversed by raising the concentration of Brf1 or Bdp1. The data support a model in which hyperphosphorylation disrupts TFIIIB during mitosis, compromising its ability to support transcription. Introduction Nuclear transcription is repressed during mitosis (reviewed by Gottesfeld and Forbes, 1997). An important step towards characterizing this regulation came with the discovery that mitotic repression can be reproduced in vitro using extracts prepared from synchronized cells. Hartl et al. (1993) demonstrated that RNA polymerase (pol) III transcription is inhibited substantially when Xenopus egg extracts are shifted into mitosis by addition of cyclin B. Although it had been postulated that chromatin condensation might be responsible for mitotic repression, pre-incubation with non-specific DNA to titrate out histones made no difference to the extent of inhibition (Hartl et al., 1993). The topoisomerase II inhibitor VM26, which blocks mitotic chromosome condensation, also failed to prevent repression (Hartl et al., 1993). Therefore, transcriptional inhibition during mitosis may not require condensed chromatin. In support of this, immunofluorescent and electron spectroscopic imaging has shown that pol II transcription of viral genes can be repressed at mitosis in the absence of chromatin condensation (Spencer et al., 2000). Cyclin-dependent kinases (cdks) from mitotic frog extracts inhibit expression of pol III templates, an effect that is blocked by the kinase inhibitor 6-dimethylaminopurine (DMAP) (Hartl et al., 1993; Gottesfeld et al., 1994; Leresche et al., 1996). In contrast, cdks purified from interphase extracts do not inhibit transcription (Hartl et al., 1993; Gottesfeld et al., 1994). Gottesfeld et al. (1994) showed that cdc2–cyclin B is sufficient to repress expression of a Xenopus 5S rRNA gene. Although this implicated cdc2–cyclin B in pol III repression, removal of cdc2 from metaphase-arrested Xenopus egg extracts does not prevent the DMAP-sensitive inhibition of a tRNA gene (Hartl et al., 1993). Furthermore, cdc2-depleted mitotic extract can inhibit transcription when mixed with interphase extract (Hartl et al., 1993). The authors concluded that mitotic extracts contain at least one other kinase besides cdc2 that is capable of repressing pol III transcription. TFIIIB is responsible for recruiting pol III to the initiation site of its templates (Kassavetis et al., 1990). It is a complex containing the TATA-binding protein (TBP) and two TBP-associated factors (TAFs) called Bdp1 and Brf1; the latter bears homology to the pol II-specific factor TFIIB (reviewed by Schramm and Hernandez, 2002). At most pol III promoters, TFIIIB is recruited by protein–protein interactions with the assembly factor TFIIIC, that binds downstream of the initiation site (reviewed by Geiduschek and Kassavetis, 2001; Schramm and Hernandez, 2002; White, 2002). Gottesfeld et al. (1994) showed that repression of Xenopus pol III transcription by cdc2 can be reversed by addition of highly purified TFIIIB, whereas TFIIIC has no effect. Although TFIIIB isolated from interphase extracts is highly efficient in this respect, TFIIIB from mitotic extracts is inactive, unless it is pre-treated with alkaline phosphatase (Gottesfeld et al., 1994). Furthermore, affinity-purified interphase TFIIIB can be inactivated using cdc2 (Gottesfeld et al., 1994). The data indicate that repression of pol III transcription in metaphase frog eggs involves inactivation of TFIIIB through the redundant action of cdc2 and one or more additional mitotic kinase. Kinases isolated from mitotic extracts using p13suc1–beads phosphorylate polypeptides of 33–34, 42 and 90–92 kDa that are present in affinity-purified TFIIIB fractions (Leresche et al., 1996). The 33 kDa species co-migrates with Xenopus TBP (Leresche et al., 1996). Furthermore, recombinant TBP can be phosphorylated in vitro using mitotic kinases, but not with purified cdc2 (Gottesfeld et al., 1994). The 92 kDa species is the same size as a TAF present in Xenopus TFIIIB (McBryant et al., 1996). Whereas the cell cycle in early Xenopus embryos consists of a simple fluctuation between S and M phases, somatic cells undergo a more complex cycle that involves sequential passage through G1, S, G2 and M. Nevertheless, mitotic HeLa cells repress pol III transcription by a mechanism that resembles the situation in frog eggs (White et al., 1995b). Thus, TFIIIB is inactivated specifically in extracts prepared from HeLa cells synchronized in M phase (White et al., 1995b). TBP becomes hyperphosphorylated during mitosis, but this does not account for the reduction in pol III transcription, since expression cannot be restored by the addition of recombinant TBP to mitotic extracts (White et al., 1995b). In contrast, affinity-purified TFIIIB TAFs reverse repression efficiently, and can restore transcription in M phase extracts (White et al., 1995b). Direct assays confirmed that the TAF component of TFIIIB is inactivated at mitosis (White et al., 1995b), but vertebrate pol III TAF sequences had not been cloned when that study was conducted and so reagents were not available to allow more precise analysis. Extensive purification of human TFIIIB has since allowed cloning of cDNAs that encode Brf1 (Wang and Roeder, 1995; Mital et al., 1996). Human Bdp1 cDNAs were isolated subsequently on the basis of homology with the yeast sequence (Schramm et al., 2000). Although Brf1 makes direct contacts with both TFIIIC and pol III (Geiduschek and Kassavetis, 2001; Schramm and Hernandez, 2002; White, 2002), Bdp1 is also necessary for productive polymerase recruitment and initiation at supercoiled class III genes (Kassavetis et al., 2001). Now that these human TAFs are available for molecular analysis, we have investigated in more detail how TFIIIB behaves at mitosis. We find that Brf1 is hyperphosphorylated when HeLa cells are arrested in metaphase, but remains associated with template DNA. In contrast, Bdp1 is released from promoters, which precludes the productive recruitment of pol III. We propose a model in which hyperphosphorylation of Brf1 at mitosis is associated with disruption of TFIIIB and release of one of its essential subunits. Results Pol III transcriptional activity in extracts of mitotic HeLa cells is determined by a balance between kinases and phosphatases Cycling HeLa cells were arrested in metaphase by treatment with nocodozole. Extracts of these mitotic cells were compared with extracts of asynchronous cells, which are predominantly (96–98%) in interphase (Terasima and Tolmach, 1963). As expected, extracts of mitotic HeLa cells are severely compromised in their ability to support transcription by pol III (Figure 1A). Figure 1.Mitotic repression of pol III transcription is mediated by phosphorylation. (A) Class III genes are transcribed more efficiently by asynchronous extracts than by mitotic extracts. pHu5S3.1 (lanes 1 and 2) or pVA1 (lanes 3 and 4) templates (500 ng) were transcribed using 14 μg of extract prepared from either asynchronous (lanes 1 and 3) or mitotic (lanes 2 and 4) cells. (B) Mitotic repression of pol III transcription can be relieved using a kinase inhibitor and accentuated with a phosphatase inhibitor. pGlu6 (500 ng) was transcribed using 30 μg of either asynchronous (lanes 1–3) or mitotic extract (lanes 4–6). Reactions in lanes 1 and 4 contained 2.4 mM DMAP; those in lanes 2 and 5 contained 2 μM okadaic acid. Download figure Download PowerPoint To assess the importance of protein kinases in maintaining repression in the mitotic extract, we tested the effect of adding DMAP, a nucleotide analogue that inhibits a broad range of protein kinases (Vesely et al., 1994). When added to extracts of asynchronous cells, DMAP inhibited transcription of a tRNA gene (Figure 1B, lanes 1 and 3). This is consistent with the fact that CK2 and ERK kinases stimulate pol III transcription in HeLa cells (Johnston et al., 2002; Felton-Edkins et al., 2003). However, pre-incubating DMAP with mitotic extract resulted in a substantial increase in tRNA gene expression (compare lanes 4 and 6). Indeed, in the presence of DMAP, the transcriptional activity of the mitotic extract was comparable with that of asynchronous extract (lanes 3 and 4). These results suggest that one or more kinases repress the pol III transcription apparatus at mitosis, but do not operate during interphase. The ability of DMAP to activate transcription implies the presence in mitotic extracts of protein phosphatase(s) that can dephosphorylate transcription factors once kinase activity is suppressed. To examine this possibility, we tested the effect of adding okadaic acid, a selective inhibitor of certain protein phosphatases (Cohen et al., 1990). The low level of tRNA transcription in mitotic extracts was reduced even further by okadaic acid (Figure 1B, lanes 5 and 6). This confirms that one or more phosphatases antagonizes the repressive effect of the mitotic kinase(s). In contrast, okadaic acid did not depress transcription when added to asynchronous extracts (lanes 2 and 3). We conclude that the activity of the pol III transcription apparatus is controlled in mitotic HeLa cells by a balance between kinase and phosphatase activities. Cdc2 activity is not required to maintain repression of pol III transcription in extracts of mitotic HeLa cells The ability of DMAP to restore pol III activity in a mitotic HeLa cell extract (Figure 1B) demonstrates that one or more protein kinases are required to maintain repression. Since the mitotic kinase cdc2–cyclin B can inactivate TFIIIB in Xenopus (Gottesfeld et al., 1994), we investigated whether it performs a similar function in a human cell system. For this purpose, we employed the purine analogue olomoucine, a potent inhibitor of cdc2–cyclin B (IC50 ∼7 μM) and cdc2–cyclin A (IC50 ∼50 μM) that is ineffective against most kinases that are not cdks (Vesely et al., 1994). Olomoucine caused no activation of tRNA synthesis when pre-incubated with a mitotic extract, even at a final concentration of 500 μM (Figure 2A). In contrast, the same concentration of DMAP produced a strong increase in tRNA expression, even though DMAP is much less effective than olomoucine against cdc2–cyclin B (IC50 ∼120 μM) (Vesely et al., 1994). To confirm the efficacy of olomoucine in our system, we tested its ability to inhibit phosphorylation of histone H1, a well-documented substrate of cdc2–cyclin B (Figure 2B). As expected, mitotic extracts contain a strong H1 kinase activity that is not seen in asynchronous extracts (compare lanes 1 and 4); this activity is inhibited efficiently by olomoucine concentrations that cause no increase in tRNA expression (lanes 3 and 4). We conclude that the mitotic repression of pol III transcription is maintained even if cdc2 is inhibited. Figure 2.Cyclin B-dependent kinases are not required to maintain mitotic repression of human pol III transcription. (A) Mitotic repression of pol III transcription can be relieved using DMAP but not olomoucine. pGlu6 (500 ng) was transcribed using 25 μg of mitotic extract that had been pre-incubated for 10 min at 30°C in the presence of buffer alone (lane 1), 0.5 mM olomoucine (lanes 2 and 3), 0.5 mM DMAP (lane 4) or 3 mM DMAP (lane 5). (B) Mitotic H1 kinase activity is inhibited efficiently by 0.5 mM olomoucine. Asynchronous (lanes 1 and 2) or mitotic (lanes 3–5) extracts (10 μg) were incubated for 10 min at 30°C in the presence of [γ-32P]ATP. Histone H1 (200 ng) was included in lanes 1–4. Olomoucine (0.5 mM) was included in lanes 2 and 3. (C) Recombinant cdc2–cyclin B has little effect on VA1 transcription in HeLa extract. Extract of asynchronous HeLa cells (10 μg) was incubated for 15 min at 30°C in the presence of 0.5 mM ATP and either buffer (lane 1), or 1 and 2 μl of baculovirus-expressed cdc2–cyclin B (lanes 2 and 3, respectively). Transcription was then started by addition of pVA1 (250 ng) and nucleotides. (D) Cdc2–cyclin B purified from mitotic HeLa cells has little effect on VA1 transcription using partially purified factors. CHep-1.0 (2.5 μl) and DE-1.0 (2.5 μl) fractions were incubated for 15 min at 30°C in the presence of 0.6 mM ATP, 4 mM vanadate and either buffer (lane 1), or 1 and 3 μl of immunopurified cyclin B-dependent kinase (lanes 2 and 3, respectively). Transcription was then started by addition of pVA1 (250 ng) and nucleotides. Download figure Download PowerPoint The apparent lack of a requirement for cdc2–cyclin B to maintain inhibition in a mitotic extract may reflect redundancy in the control mechanisms. We therefore tested directly whether cdc2–cyclin B will repress pol III transcription in HeLa extracts. Baculovirus-expressed recombinant cdc2–cyclin B was pre-incubated with asynchronous extract in the presence of ATP; after 15 min, we added nucleotides and template and then assayed for transcription (Figure 2C). Inclusion of cdc2–cyclin B made little difference to the level of VA1 expression. Since asynchronous extract might contain activities which antagonize the action of cdc2–cyclin B, we repeated these experiments using extracts of mitotic cells, but again the kinase had little effect (data not shown). We also tested cdc2–cyclin B that had been immunopurified from mitotic HeLa cells. As with the recombinant protein, this kinase had little effect on pol III output when added to crude extract. It was also used in a reconstituted system composed of partially purified transcription factors, but again failed to inhibit expression (Figure 2D). Several preparations of recombinant and natural cdc2–cyclin B were tested and the kinase activity of these was confirmed using histone H1 as substrate. Some kinase preparations produced weak inhibitory effects that did not require ATP and could not be blocked by DMAP; this was presumed to be caused by contaminants. However, we have been unable to detect any consistent response to added cdc2–cyclin B that is dependent on kinase activity. The inhibitory effect of mitotic kinase(s) on pol III transcription is antagonized specifically by PP2A As the phosphatase inhibitor okadaic acid depresses the level of tRNA synthesis in mitotic extracts (Figure 1B), one or more endogenous phosphatases must antagonize the repressive effect of the kinase(s) present at metaphase. We adopted a pharmacological approach to identify the phosphatase(s) responsible for this effect. In Figure 1B, we used a concentration of okadaic acid (2.5 μM) that will inhibit a range of protein phosphatases (Cohen et al., 1990). This decreased tRNA production by ∼2-fold when added to mitotic extract. When used in the nanomolar range, okadaic acid displays greater specificity for the PP1 and PP2A families of serine/threonine phosphatases (Cohen et al., 1990). We found that 100 nM okadaic acid was just as effective at depressing tRNA synthesis, again producing an ∼2-fold decrease (Figure 3A, lanes 1 and 2). Indeed, a slight reduction (1.3-fold) was seen with only 0.2 nM okadaic acid (lane 3). We also tested the unrelated toxin calyculin A, another potent inhibitor of PP1 and PP2A (Cohen et al., 1990). Calyculin A produced a 2-fold decrease in transcription of the tRNA gene at 100 nM (lane 4) and a 1.5-fold decrease at 0.2 nM (lane 5). These results suggest that PP1 and/or PP2A may counteract repression of TFIIIB in mitotic HeLa cells. Figure 3.PP2A inhibitors depress pol III transcription in extracts of mitotic HeLa cells. (A) Mitotic repression of pol III transcription can be accentuated using okadaic acid or calyculin A. pGlu6 (500 ng) was transcribed using 25 μg of mitotic extract that had been pre-incubated for 10 min at 30°C in the presence of buffer alone (lane 1), 100 or 0.2 nM okadaic acid (lanes 2 and 3, respectively), and 100 or 0.2 nM calyculin A (lanes 4 and 5, respectively). (B) PP2A inhibitor polypeptide depresses pol III transcription in extracts of mitotic HeLa cells. pVAI (250 ng) was transcribed with 25 μg of mitotic extract pre- incubated for 10 min at 30°C in the presence of buffer alone (lanes 1 and 4), and 25 or 100 ng of I1PP2A (lanes 2 and 3, respectively). Download figure Download PowerPoint Whereas PP2A displays a similar sensitivity to okadaic acid and calyculin A, PP1 is 10- to 100-fold more sensitive to the latter (Cohen et al., 1990). The fact that these toxins were equally potent in reducing tRNA synthesis by a mitotic extract (Figure 3A) suggests that the phosphatase responsible is PP2A rather than PP1. To confirm this, we tested I1PP2A, a polypeptide inhibitor with high potency and apparently absolute specificity for PP2A (Li et al., 1995). This decreased pol III transcription efficiently when added to mitotic extract (Figure 3B). These results implicate endogenous PP2A as being responsible for antagonizing the inhibitory effect on TFIIIB of kinase(s) active in metaphase-arrested HeLa cells. The TFIIIB-specific TAF Brf1 is hyperphosphorylated during mitosis It was found previously that repression of pol III transcription in mitotic extracts can be reversed by titration of affinity-purified fractions containing TFIIIB TAFs, whereas TFIIIC, pol III or recombinant TBP do not restore expression (White et al., 1995b). This led to the model that mitotic repression is mediated through specific inactivation of one or more of the TAF subunits of TFIIIB (White et al., 1995b). Further characterization was not possible at that time, as the TAF components of human TFIIIB had not been identified. However, it is now established that the TAF component of human TFIIIB comprises the essential subunits Brf1 and Bdp1 (Schramm and Hernandez, 2002). As the results above suggest that repression is mediated through phosphorylation, we used immunoblotting to look for changes in the electrophoretic mobility of the TFIIIB TAFs, that might result from addition of phosphate groups. Although the mobility of Bdp1 remains constant after entry into mitosis, Brf1 in mitotic extracts migrates more slowly than Brf1 in asynchronous cell extracts (Figure 4A). That this shift in mobility is due to phosphorylation was suggested by using kinase and phosphatase inhibitors (Figure 4B). Pre- incubation with DMAP increased the mobility of mitotic Brf1 (lane 2), whereas inclusion of okadaic acid had the opposite effect, accentuating the slow migrating forms of mitotic Brf1 (lane 3). Thus, kinase and phosphatase inhibitors that alter the activity of the pol III transcription apparatus have a parallel effect on the electrophoretic mobility of Brf1. Figure 4.The Brf1 subunit of TFIIIB is hyperphosphorylated during mitosis. (A) The electrophoretic mobility of Brf1 decreases at mitosis. Asynchronous (lane 1) and mitotic (lane 2) extracts (50 μg) were resolved on a 7.8% SDS–polyacrylamide gel and analysed by western immunoblotting with anti-Bdp1 antibody 2663 (upper panel) and anti-Brf1 antibody 330 (lower panel). (B) The altered mobility of Brf1 at mitosis is due to phosphorylation. Extracts (30 μg) of mitotic (lanes 1–3) or asynchronous (lane 4) cells were incubated for 35 min at 30°C in the presence of 0.5 mM ATP. Reactions shown in lanes 2 and 3 also contained 2.4 mM DMAP or 2 μM okadaic acid, respectively. Samples were resolved on a 10% SDS–polyacrylamide gel and immunoblotted with anti-Brf1 antibody CSH409. (C) Brf1 is hyperphosphorylated in metaphase-arrested cells. HeLa cells were transiently transfected with pcDNA3HA.BRF (10 μg) and then synchronized in S or M phases and labelled with [32P]orthophosphate for 3 h prior to harvesting. Proteins immunoprecipitated with anti-HA antibody F-7 (lanes 1 and 2) or non-immune serum (lanes 3 and 4) were resolved on a 7.8% SDS–polyacrylamide gel, transferred to nitrocellulose and then visualized by autoradiography (top) and western blotting with F-7 (bottom). Download figure Download PowerPoint In vivo labelling was used to test if Brf1 becomes hyperphosphorylated at mitosis in living cells. HeLa cells were transfected with haemagglutinin (HA)-tagged Brf1, synchronized in S or M phases and then labelled with [32P]orthophosphate; S phase synchronization was used to remove the ∼2% of mitotic cells that are present in an asynchronous population. Protein immunoprecipitated using either anti-HA antibody or a pre-immune control was resolved on a denaturing gel and subjected to autoradiography and western blotting. Although comparable amounts of Brf1 were immunoprecipitated from the two cell populations, phosphate labelling was ∼6-fold higher in the mitotic cells (Figure 4C). Specificity was confirmed by the absence of Brf1 in the control immunoprecipitates. We conclude that Brf1 is hyperphosphorylated by one or more kinases that become active at metaphase. Promoter occupancy by TFIIIB is compromised during mitosis Mitotic phosphorylation has been shown to dissociate several transcription factors from condensing chromosomes, such as Oct-1, Sp1 and Ikaros (Segil et al., 1991; Martinez-Balbas et al., 1995; Dovat et al., 2002). We therefore investigated whether TFIIIB might also be released in this way. To this end, chromatin immunoprecipitation (ChIP) assays were used to compare promoter occupancy between asynchronous and mitotic cells in vivo (Figure 5). As expected, TFIIIC2, TFIIIB and pol III were all detected at 5S rRNA and tRNA genes, whereas little or no Oct-1 or TFIIB was found at these sites. Antibodies against the individual subunits of TFIIIB revealed a slight reduction (∼20%) in the amounts of TBP and Brf1 associated with promoters in mitotic cells compared with asynchronous populations. However, occupancy of the third TFIIIB subunit, Bdp1, was more substantially diminished. Levels of bound pol III paralleled those of Bdp1, consistent with the fact that Bdp1 is necessary for efficient recruitment of pol III onto templates (Kassavetis et al., 2001). In contrast to these results, TFIIIC2 occupancy was undiminished at mitosis. Indeed, we reproducibly observed an increase in TFIIIC2 signals in mitotic cells. Although this might reflect increased binding, it might also result from an increase in antibody accessibility due to diminished association of pol III and TFIIIB. Whatever the cause, it confirms that the observed reductions in occupancy by TFIIIB and pol III are specific. The data suggest that Bdp1 and pol III are preferentially released from mitotic chromatin. Figure 5.Promoter occupancy by TFIIIB is compromised during mitosis. (A) ChIP assay showing levels of Oct-1, TFIIB, TBP, Brf1, Bdp1, pol III (BN51 subunit) and TFIIIC2 (TFIIIC110 subunit) associated with 5S rRNA and tRNAArg genes in asynchronous (A) or mitotic (M) HeLa cells, as indicated. Serial dilutions of input DNA confirm that PCRs are within the linear range and that A and M samples utilize equivalent amounts of input. (B) Quantitation of ChIP assays. PCR products from three independent ChIP experiments were quantified for 5S rRNA (top) and tRNAArg genes (bottom); after normalization to input, the mean and standard deviations are shown for the ratio of mitotic/asynchronous signals. Download figure Download PowerPoint As an independent test of this, purified metaphase chromosome preparations were examined by immunoblotting. As reported (Segil et al., 1991), Oct-1 was barely detectable in the metaphase chromosomes, showing that they are relatively free of contamination (Figure 6). In contrast, TBP was readily detected, consistent with previous evidence that some TFIID remains tightly associated with condensed mitotic chromosomes (Segil et al., 1996; Christova and Oelgeschläger, 2001; Chen et al., 2002). Similarly, immunoblotting revealed the presence of Brf1, as shown previously (Chen et al., 2002). A substantial proportion of this chromosomally associated Brf1 was found to migrate more slowly than the Brf1 present in asynchronous cells, consistent with its reduced mobility in mitotic extracts (Figure 4A). Some faster migrating Brf1 was also detected in the metaphase chromosomes; it is unclear whether this reflects the existence of distinct forms in vivo, or partial dephosphorylation during harvesting. Nevertheless, these observations suggest that hyperphosphorylation does not release Brf1 from chromosomes during M phase. In contrast to TBP and Brf1, the third TFIIIB subunit, Bdp1, was barely detectable in the same chromosome preparations (Figure 6). This was also true of RPC155, the largest subunit of pol III. However, TFIIIC220, the DNA-binding subunit of TFIIIC2, was found associated with the purified metaphase chromosomes (Figure 6). These observations therefore support the ChIP data and point to a differential displacement from mitotic chromatin of components of the pol III machinery, with Bdp1 and pol III being released preferentially. Figure 6.TBP and Brf1 remain associated with purified metaphase chromosomes, whereas Bdp1 is not detectable. Metaphase chromosomes (50 μl; lane 1) and asynchronous cell extracts (50 μg; lane 2) were resolved on a 7.8% SDS–polyacrylamide gel and then analysed by western immunoblotting with anti-TBP antibody 58C9, anti-Brf1 antibody 128, anti-Bdp1 antibody 2663, anti-pol III antibody 1900, anti-TFIIIC220 antibody Ab2 and anti-Oct-1 antibody C-21, as indicated. Download figure Download PowerPoint The affinity of Brf1 for Bdp1 may be diminished during mitosis On TATA-less promoters such as 5S rRNA and tRNAArg, TFIIIB is recruited through binding of Brf1 to DNA-bound TFIIIC2; the presence of Bdp1 in turn depends on its interaction with Brf1 (Geiduschek and Kassavetis, 2001; Schramm and Hernandez, 2002). The mitotic release of Bdp1 from these genes suggests that its affinity for Brf1 may be diminished during this phase of the cell cycle. To test this, we carried out co-immunoprecipitations to monitor the Brf1–Bdp1 interaction. Brf1 was labelled with 35S by translation in vitro and then mixed with extract of asynchronous or metaphase-arrested HeLa cells. Although not apparent under the running conditions used for Figure 7, a differential shift in electrophoretic mobility indicates that the exogenous Brf1 becomes hyperphosphorylated in the mitotic extract (data not shown). With asynchronous extracts, antiserum against Bdp1 was able to co-immunoprecipitate Brf1, but with mitotic extracts this was reduced to the background levels observed with pre-immune serum (Figure 7A). In contrast, similar amounts of Brf1 were co-immunoprecipitated with TBP from mitotic and asynchronous extracts (Figure 7B). These observations suggest that the interaction between Bdp1 and Brf1 may be selectively destabilized at M phase. This might explain the observed release of Bdp1 from metaphase chromosomes. Figure 7.The Brf1–Bdp1 interaction may be compromised specifically at mitosis. (A) Reticulocyte lysate (10 μl) containing in vitro-translated Brf1 was mixed with extract (150 μg) of asynchronous (lanes 2 and 4) or metaphase-arrested HeLa cells (lanes 3 and 5) prior to immunoprecipitation with anti-Bdp1 antiserum (lanes 2 and 3) or pre-immune serum (lanes 4 and 5). Protein" @default.
• W1969606627 created "2016-06-24" @default.
• W1969606627 creator A5007470711 @default.
• W1969606627 creator A5024647268 @default.
• W1969606627 creator A5067344213 @default.
• W1969606627 date "2003-11-03" @default.
• W1969606627 modified "2023-10-18" @default.
• W1969606627 title "TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo" @default.
• W1969606627 cites W1531234455 @default.
• W1969606627 cites W1750653145 @default.
• W1969606627 cites W1856072495 @default.
• W1969606627 cites W1928732831 @default.
• W1969606627 cites W1970446592 @default.
• W1969606627 cites W1981593440 @default.
• W1969606627 cites W1997850825 @default.
• W1969606627 cites W2006630070 @default.
• W1969606627 cites W2009709163 @default.
• W1969606627 cites W2015020895 @default.
• W1969606627 cites W2017050402 @default.
• W1969606627 cites W2025375270 @default.
• W1969606627 cites W2036733966 @default.
• W1969606627 cites W2038013843 @default.
• W1969606627 cites W2040690000 @default.
• W1969606627 cites W2041108171 @default.
• W1969606627 cites W2043848225 @default.
• W1969606627 cites W2051816569 @default.
• W1969606627 cites W2053043602 @default.
• W1969606627 cites W2058311130 @default.
• W1969606627 cites W2059198116 @default.
• W1969606627 cites W2061857365 @default.
• W1969606627 cites W2073932060 @default.
• W1969606627 cites W2080927800 @default.
• W1969606627 cites W2082395806 @default.
• W1969606627 cites W2089528428 @default.
• W1969606627 cites W2091702992 @default.
• W1969606627 cites W2094401820 @default.
• W1969606627 cites W2097303043 @default.
• W1969606627 cites W2101095087 @default.
• W1969606627 cites W2110640745 @default.
• W1969606627 cites W2116766341 @default.
• W1969606627 cites W2118751823 @default.
• W1969606627 cites W2119361029 @default.
• W1969606627 cites W2129303921 @default.
• W1969606627 cites W2134388771 @default.
• W1969606627 cites W2139323446 @default.
• W1969606627 cites W2140195900 @default.
• W1969606627 cites W2152685009 @default.
• W1969606627 cites W2161879504 @default.
• W1969606627 cites W2163491295 @default.
• W1969606627 cites W2166572022 @default.
• W1969606627 doi "https://doi.org/10.1093/emboj/cdg544" @default.
• W1969606627 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/275401" @default.
• W1969606627 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14592981" @default.
• W1969606627 hasPublicationYear "2003" @default.
• W1969606627 type Work @default.
• W1969606627 sameAs 1969606627 @default.
• W1969606627 citedByCount "63" @default.
• W1969606627 countsByYear W19696066272012 @default.
• W1969606627 countsByYear W19696066272013 @default.
• W1969606627 countsByYear W19696066272014 @default.
• W1969606627 countsByYear W19696066272015 @default.
• W1969606627 countsByYear W19696066272017 @default.
• W1969606627 countsByYear W19696066272018 @default.
• W1969606627 countsByYear W19696066272019 @default.
• W1969606627 countsByYear W19696066272021 @default.
• W1969606627 countsByYear W19696066272022 @default.
• W1969606627 crossrefType "journal-article" @default.
• W1969606627 hasAuthorship W1969606627A5007470711 @default.
• W1969606627 hasAuthorship W1969606627A5024647268 @default.
• W1969606627 hasAuthorship W1969606627A5067344213 @default.
• W1969606627 hasBestOaLocation W19696066272 @default.
• W1969606627 hasConcept C101762097 @default.
• W1969606627 hasConcept C104317684 @default.
• W1969606627 hasConcept C11960822 @default.
• W1969606627 hasConcept C150194340 @default.
• W1969606627 hasConcept C153957851 @default.
• W1969606627 hasConcept C207001950 @default.
• W1969606627 hasConcept C54355233 @default.
• W1969606627 hasConcept C67705224 @default.
• W1969606627 hasConcept C86803240 @default.
• W1969606627 hasConcept C93304396 @default.
• W1969606627 hasConcept C95444343 @default.
• W1969606627 hasConceptScore W1969606627C101762097 @default.
• W1969606627 hasConceptScore W1969606627C104317684 @default.
• W1969606627 hasConceptScore W1969606627C11960822 @default.
• W1969606627 hasConceptScore W1969606627C150194340 @default.
• W1969606627 hasConceptScore W1969606627C153957851 @default.
• W1969606627 hasConceptScore W1969606627C207001950 @default.
• W1969606627 hasConceptScore W1969606627C54355233 @default.
• W1969606627 hasConceptScore W1969606627C67705224 @default.
• W1969606627 hasConceptScore W1969606627C86803240 @default.
• W1969606627 hasConceptScore W1969606627C93304396 @default.
• W1969606627 hasConceptScore W1969606627C95444343 @default.
• W1969606627 hasIssue "21" @default.
• W1969606627 hasLocation W19696066271 @default.
• W1969606627 hasLocation W19696066272 @default.
• W1969606627 hasLocation W19696066273 @default.
• W1969606627 hasLocation W19696066274 @default.

• next