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J. Biol. Chem., Vol. 279, Issue 15, 15339-15347, April 9, 2004
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¶
From the
Section of Developmental and Stem Cell Biology, Joslin Diabetes Center, and the
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, September 29, 2003 , and in revised form, January 13, 2004.
| ABSTRACT |
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| INTRODUCTION |
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The general transcription factor TFIID is of particular interest because it establishes the start site and provides enzymatic activities that may regulate transcription (5, 6). TFIID is comprised of the TATA-binding protein (TBP) along with
14 TBP-associated factors (TAFIIs). TAFIIs interact with core promoter elements and contact a diverse array of upstream transactivators (5-8). TAF-1 and TAF-2 together bind directly to the initiator (Inr) element, which encompasses the start site (9). TAF-1, the largest TAFII, is also necessary for TFIID stability and possesses histone acetyltransferase, kinase, and ubiquitin conjugating activities (10). TAF-1 is unique to TFIID, but many other TAFIIs are also found in the SPT-ADA-GCN5 (SAGA)-related complexes (5, 6), which are similar in structure to TFIID but lack TBP and contain a GCN5-related histone acetyltransferase instead of TAF-1.
In S. cerevisiae conditional mutation or shut-off analyses suggest that many individual TAFIIs have surprisingly specific functions (5, 6). For example, whole genome analyses indicate that TFIID-specific taf-1 and taf-2 are essential for expression of only 14 and 3% of genes, respectively (3, 4, 11), and chromatin immunoprecipitation has detected significant TAFII occupancy only at TFIID-dependent genes (12, 13). These studies suggest that in yeast a major proportion of transcription involves a TAFII-independent form of TFIID and that the TFIID-specific TAFIIs are each required to transcribe only a modest fraction of the genome, although this model remains a subject of investigation and debate (14). In contrast, expression of the majority of yeast genes is prevented by conditional loss of either TAF-9, which is shared between TFIID and SAGA, or of Taf-1 and the SAGA histone acetyltransferase GCN5 simultaneously, suggesting that TFIID and SAGA are redundant at many genes (4-6).
Although considerable information has been obtained about TAFII functions in yeast, it is a distinct question how TAFIIs contribute to transcription in vivo in metazoans, particularly in the context of the complex processes of tissue development or differentiation. The three-dimensional structure of TFIID is conserved among eukaryotes (15-17), predicting a similar conservation of function. However, transcription in metazoans involves a more complex interplay between promoters and long range elements, as well as additional PIC components and TAFII isoforms that are not present in yeast (2). Loss of TAFII function in metazoans has been difficult to study because TAFIIs are expressed both maternally and zygotically, thus complicating interpretation of mutant phenotypes. For example, in Drosophila taf-1 mutants have pleiotropic defects, but the consequences of eliminating both maternally and zygotically expressed TAF-1 have not been determined (18). In hamster cells a conditional taf-1 mutation decreased expression of
18% of genes and caused apoptosis (19), a finding that is consistent with yeast data but does not appear to involve complete ablation of TAF-1 function.
In the Caenorhabditis elegans embryo, it is possible to use RNA interference (RNAi) (20) to inhibit both maternal and zygotic expression of C. elegans TAFIIs. If transcription is prevented in the early C. elegans embryo, maternally supplied mRNAs maintain viability until the 100-cell stage, making it feasible to block expression of even essential transcription factors (21). Using this strategy, we determined previously that TAF-4 is required for essentially all early embryonic transcription (22). In contrast, TAF-5, TAF-9, and TAF-10 were required for significant and comparable fractions of early transcription but appeared to be dispensable at most metazoan-specific promoters (22, 23). Each of the TAFIIs we have analyzed is shared between TFIID and SAGA-like complexes, leaving open the question of how broadly TFIID is required in the embryo. This issue is of particular interest because a major fraction of C. elegans embryonic transcription requires the TBP isoform TLF(TRF2), which does not associate with TAFIIs (24-26).
In this study we have determined that TFIID-specific TAF-1 is essential for most transcription in the developing C. elegans embryo. In contrast to the shared TFIID/SAGA TAF-5, -9, or -10, TAF-1 is needed for many metazoan-specific genes to be expressed at appropriate levels. TAF-1 does not appear to be universally essential for early embryonic transcription, however, unlike TAF-4. TAF-2 appears to be required for a similarly extensive fraction of embryonic transcription as TAF-1. We have also obtained evidence for functional overlap between TAF-1 and the C. elegans CBP/p300 ortholog cbp-1. We conclude that in the early C. elegans embryo TFIID and promoter recognition by TAFIIs are important for transcription of most genes, including many that require TLF.
| EXPERIMENTAL PROCEDURES |
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Immunostaining and Fluorescence AnalysisRabbit antisera were raised against the TAF-1 peptide VSQKPHKDENATPVPVKKLVT with an N-terminal Cys added and affinity purified (22). For TAF-1 staining, embryos were fixed with 1% paraformaldehyde and 0.1% glutaraldehyde before treating with methanol. Washes and antibody incubations were performed in PBT (1x phosphate-buffered saline, 1% Triton X-100, 1% bovine serum albumin) prior to staining. TAF-1 antibody staining was competed by the cognate but not heterologous peptides (not shown). Staining with
-TAF-9,
-TAF-10,
-pol II (pol 3/3) (22), P-CTD (anti-phospho-Ser-5) (28), H5 (anti-phospho-Ser-2) (Covance), and CBP-1 (29) was performed as in Ref. 22.
-TBP-1 and
-TLF-1 immunostaining was performed as in Ref. 25. Staining with the H14 antibody was performed as for H5 and provided results identical to those with the P-CTD antibody. Green fluorescent protein (GFP) analysis, image capture, and manipulation were performed as described by Walker et al. (22).
RNAi AnalysisFor injection of dsRNA, cDNA fragments for taf-1 (nucleotides 3066-3942 and 4329-4936) and taf-2 (nucleotides 385-1347) were generated by PCR from a C. elegans cDNA library (gift of Marc Vidal). Identical results were obtained from both taf-1 cDNAs as well as from a taf-1 clone (yk6h7) obtained from Yuji Kohara (NIG, Japan). dsRNA synthesis was synthesized in vitro with Megascript (Ambion). Injection and analysis of embryos were performed as described by Walker et al. (22). Simultaneous double RNAi was performed with a 1:1 mixture of dsRNAs. In parallel, a 1:1 dilution of each individual dsRNA with an unrelated dsRNA (glp-1) resulted in appropriate terminal arrest, reporter gene expression, and CTD phosphorylation levels (not shown). For feeding of dsRNA, cDNA fragments for taf-1 (nucleotides 2791-3408) and ama-1 (nucleotides 1254-2259) were inserted into the feeding vector pPD129.36 (gift of Andy Fire). Synchronized L4 larvae were placed on bacteria expressing dsRNA to gfp (pPD128.110, gift of Andy Fire), ama-1, or taf-1 for 36 h. taf-1 and ama-1(RNAi) embryos produced from feeding dsRNA at 36 h had anti-phospho-Ser-2 staining patterns and LET-858::GFP expression similar to injected dsRNA at 24 h (not shown).
RT-PCRN2 hermaphrodites were fed dsRNA for taf-1, ama-1, or gfp (control). Adults were washed five times in phosphate-buffered saline, and embryos were collected by bleaching. After lysing embryos in 0.5% SDS, 5%
-mercaptoethanol, 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, and 0.5 mg/ml proteinase K (Invitrogen), RNA was extracted with Tri-Reagent (Sigma). cDNA was produced from 1 µg of control RNA and from equivalent numbers of ama-1 or taf-1(RNAi) embryos (Superscript II, Invitrogen). PCR was performed using HotMix (Eppendorf). Each primer set was tested on cDNA produced from at least two independent RNA preparations. At least three dilutions of cDNA were tested, and multiple cycle numbers were used to assure linearity of reaction. Primers were designed to span at least one intron (sequences available upon request).
Immunoblot AnalysisControl, taf-1(RNAi), or ama-1(RNAi) embryos from feeding were collected as for RT-PCR, then lysed by sonication in 100 mM Tris, pH 7.9, 3 mM MgCl2, 0.3 M KCl, 0.1% Nonidet P-40, 1 mM dithiothreitol, and 20% glycerol. Proteins were separated on 6.5% gels, transferred to nitrocellulose, and probed with the antibodies indicated in Fig. 5. In this experiment anti-phospho-Ser-5 was H14 and
-pol II was ARNA3 (Research Diagnostics). Secondary antibodies used were goat anti-rabbit IgM (Kirkegaard and Perry Laboratories) for anti-phospho-Ser-5 and anti-phospho-Ser-2, goat anti-mouse (Jackson Immunologicals) for
-pol II, and goat anti-rabbit (Jackson Immunologicals) for
-CBP-1. Blots were visualized by enhanced chemiluminescence (Amersham Biosciences).
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| RESULTS |
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Severely Reduced pol II CTD Phosphorylation Levels in taf-1(RNAi) EmbryosWe investigated how mRNA transcription was affected in taf-1(RNAi) embryos first by analyzing phosphorylation of the pol II large subunit CTD. The CTD consists of multiple repeats that are based upon the consensus YSPTSPS (33). Polymerase II is initially recruited in an unphosphorylated form, then at the promoter its CTD repeat is phosphorylated on Ser-5 by the TFIIH kinase (28, 34). During elongation the distribution of CTD phosphorylation shifts to Ser-2 (34, 35), which is phosphorylated by the P-TEFb kinase (31, 36). CTD Ser-5 and Ser-2 phosphorylation can be specifically detected in C. elegans embryonic nuclei by staining with the H14 (or P-CTD) and H5 antibodies, respectively (22, 28, 37), which we refer to as anti-phospho-Ser-5 and anti-phospho-Ser-2 for clarity (Figs. 4 and 5).
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In taf-1(RNAi) embryos nucleoplasmic anti-phospho-Ser-5 and anti-phospho-Ser-2 staining was dramatically and consistently reduced in all somatic cells (Fig. 4A). The level of anti-phospho-Ser-2 staining in these RNAi embryos was only slightly higher than the background seen in transcriptionally silent ama-1(RNAi) or taf-4(RNAi) embryos and was significantly lower than in taf-5, taf-9, or taf-10(RNAi) embryos (Fig. 4A) (22, 23), suggesting that most pol II transcription had been prevented. Accordingly, in taf-1(RNAi) embryos nucleoplasmic anti-phospho-Ser-5 staining was decreased proportionally to anti-phospho-Ser-2 staining, except that two anti-phospho-Ser-5 foci like those normally present in the germ line were prominent in somatic cells (Fig. 4, A and B), as had been observed previously in taf-4, taf-5, taf-9, and taf-10(RNAi) embryos. Anti-phospho-Ser-2 and anti-phospho-Ser-5 staining levels were affected similarly when taf-1 and taf-10 were inhibited simultaneously by RNAi (taf-1; taf-10(RNAi); Fig. 4A), indicating that the residual CTD phosphorylation in taf-1(RNAi) embryos was not sensitive to depletion of an additional TAFII and was unlikely to derive from a partial RNAi effect. Significantly, in individual taf-1(RNAi) embryos CTD phosphorylation levels were proportionally decreased between the onset of transcription at the four-cell stage until terminal arrest (data not shown), suggesting that this reduction derived from a continuous broad decrease in pol II transcription and not a stage-specific abnormality.
The conclusions of these antibody staining experiments were supported by immunoblot analyses of embryo extracts, which demonstrated that in taf-1(RNAi) embryo populations CTD Ser-2 and Ser-5 phosphorylation was only barely detectable (Fig. 5). In contrast, and also consistent with immunofluorescence experiments (Fig. 2), the levels of total pol II present in taf-1(RNAi) embryos were at least equivalent to the levels detected in control extracts (
-pol II, Fig. 5). The dramatic decreases in pol II CTD phosphorylation which accompanied taf-1 RNAi knock-down indicated that taf-1 is required for the majority of pol II transcription in the early embryo.
Decreased Expression of Conserved and Metazoan-specific Genes in taf-1(RNAi) EmbryosTo evaluate the importance of TAF-1 for expression of individual genes in vivo, we used two types of assay. First, we used RNAi to inhibit taf-1 expression in C. elegans strains that carry transgenic reporter genes. These transgenes include intact regulatory regions fused to GFP and are expressed in parallel to the corresponding endogenous genes. A unique advantage of this system is that it allows analysis of de novo gene expression in individual living embryos. Each of these reporters is fully dependent upon taf-4, but in taf-5, taf-9, and taf-10(RNAi) embryos the metazoan-specific reporters we have analyzed are expressed at WT levels (22, 23). Second, we used RT-PCR to measure the expression of endogenous genes in control and RNAi embryos.
We first investigated the expression of two groups of genes that are expressed widely within the embryo. rps-5, let-858, and the heat shock gene hsp-16.2 each has orthologs in unicellular eukaryotes as well as in metazoans. In C. elegans, their expression requires taf-5, taf-9, and taf-10 in addition to taf-4 (22, 23), and in yeast expression of rps-5 and other ribosomal protein genes is dependent upon many TAFIIs (7, 11). Expression of the corresponding GFP reporters was abolished in taf-1(RNAi) embryos (Fig. 6A). Accordingly, levels of endogenous rps-5 and rps-26 mRNA were also lower in ama-1 and taf-1(RNAi) embryos (Fig. 6B). The residual rps-5 and rps-26 mRNA that was detected in ama-1 and taf-1(RNAi) embryos is likely to be derived from the previously described maternal expression of these genes (39, 40), which would not be affected in our assays. In addition, and in contrast to TAF-5, -9, and -10, TAF-1 was also critical for expression of the widely expressed metazoan-specific genes cki-2 (CDK inhibitor) and sur-5 (mitogen-activated protein kinase kinase pathway) (Table I and Fig. 6B).
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70% of taf-1(RNAi) embryos, however (Fig. 7C and, Table I), a difference that may derive from the multiple inputs that act at this promoter (Fig. 7A). The robustness of this end-1 expression suggests that the reductions in transcription of other genes seen in taf-1(RNAi) embryos reflects a requirement for TAF-1 at those genes and not a nonspecific abnormality.
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This broad requirement for a TFIID-specific TAFII in the early embryo is surprising because in C. elegans most embryonic transcription involves the TBP isoform TLF(TRF2) (24, 25), which does not associate with TAFIIs (26). In Xenopus and zebrafish embryos TLF and TBP are required at partially overlapping sets of genes (45, 46). In Drosophila cells TLF and TFIID can direct initiation from different promoter types, however (26), and in mice TLF is required specifically at spermiogenesis genes (47), suggesting that TLF is a promoter specificity factor that may act at different genes from TFIID. However, C. elegans TLF is needed for appropriate expression of pha-4 (24, 25), which also requires TAF-1 (Figs. 6B and 7C). To investigate this question further, we examined expression of pes-10, which is activated when embryonic transcription begins and is TLF-dependent and bound by TLF in vivo (25). In taf-1(RNAi) embryos TLF was present (Fig. 2), but our analysis showed that pes-10 expression was decreased dramatically (Fig. 6B and Table I). Significantly, TLF was also required at rps-5 and let-858 (Fig. 6, A and B), genes that are taf-1-dependent and conserved in yeast, which lack TLF. We conclude that in the embryo transcription of various individual genes involves both TLF and TFIID.
Overlapping Requirements for TAF-1 and CBP-1 at Some Metazoan-specific GenesIn S. cerevisiae TAF-1 and GCN5 appear to have redundant functions at many genes (4). Surprisingly, RNAi knock-down of the single C. elegans GCN5-related histone acetyltransferase (CeGCN5; ORF Y47G6A.6) depleted its mRNA but did not impair development and did not detectably reduce gene expression in the taf-1 RNAi background.2 This finding does not suggest major functional overlap between CeGCN-5 and TAF-1, although contributions from persistent maternal CeGCN-5 protein cannot be ruled out. Because the human SAGA-like complex TFTC can function cooperatively with the metazoan histone acetyltransferase p300/CBP (48), we tested whether TAF-1 and the C. elegans p300/CBP ortholog CBP-1 might have overlapping functions.
CBP-1 is required for all non-neuronal differentiation in C. elegans (49), but cbp-1 RNAi did not detectably reduce embryonic CTD phosphorylation levels (Fig. 4C), suggesting a minimal effect on total transcription. Accordingly, in cbp-1(RNAi) embryos many genes were expressed at near normal levels, including some that are upstream of CBP-1-dependent differentiation (med-1, pha-4, and elt-5) (Figs. 6B and 7, B and C). CTD phosphorylation levels were not distinguishably different between taf-1(RNAi) and taf-1; cbp-1(RNAi) embryos (Fig. 4C), suggesting that most TAF-1 and CBP-1 functions are nonredundant, but simultaneous interference with cbp-1 eliminated taf-1-independent med-1, pha-4, and elt-5 expression (taf-1; cbp-1(RNAi); Fig. 7, B and C). In contrast, in taf-10; cbp-1(RNAi) embryos med-1 and elt-5 were expressed at near WT levels (Fig. 7, B and C, and data not shown). We conclude that during embryonic development CBP-1 has functions that overlap with those of TAF-1, but not necessarily other TAFIIs.
Comparably Reduced CTD Phosphorylation in taf-1(RNAi) and taf-2(RNAi) EmbryosThe broad requirement for TAF-1 we have observed predicts a similarly broad role for TAF-2, which cooperates with TAF-1 to bind to the Inr (9). In yeast taf-2 is required to transcribe only 3% of the genome, however, the smallest fraction of any TAFII (4, 11). TAF-2 is TFIID-specific in yeast, but in humans it is also present within the TFTC complex, which can substitute for TFIID to initiate transcription (50). When expression of C. elegans TAF-2 (Fig. 1B) was inhibited by RNAi, embryonic development was arrested similarly to ama-1(RNAi) and taf-1(RNAi) embryos (data not shown). Significantly, at each stage taf-2(RNAi) embryos were indistinguishable from taf-1(RNAi) embryos in their anti-phospho-Ser-2 and anti-phospho-Ser-5 staining levels (Fig. 4D), indicating that TAF-1 and TAF-2 are required for similarly extensive proportions of C. elegans embryonic transcription.
| DISCUSSION |
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It appears unlikely that the limited transcription that occurred in taf-1(RNAi) embryos derived from incomplete RNAi. Expression of the conserved genes let-858, rps-5, and hsp-16.2 was decreased as severely in taf-1(RNAi) embryos as in ama-1(RNAi) embryos (Fig. 6 and data not shown). taf-1(RNAi) phenotypes were not enhanced by simultaneous inhibition of taf-10 (Figs. 4A and 7B) and were highly consistent and accompanied by depletion of TAF-1 protein (Fig. 2) and mRNA (Fig. 3D). We conclude that the residual transcription levels in taf-1(RNAi) embryos derive from a small group of largely taf-1-independent genes, including end-1, and from low level expression of metazoan-specific genes such as med-1, pha-4, and elt-5 (Figs. 6B and 7, B and C).
TAF-1 represents a third functional class of C. elegans TAFII defined by our experiments (Table II). Unlike TAF-4, TAF-1 does not appear to be generally essential for transcription. TAF-1 is distinct from the TFIID/SAGA TAFII group represented by TAF-5, -9, and -10, however, because those TAFIIs are dispensable widely at metazoan-specific genes (Figs. 6B and Fig. 7 and Table I). Accordingly, in taf-1(RNAi) embryos nucleoplasmic pol II CTD phosphorylation levels were intermediate between those found in taf-4(RNAi) and taf-5, -9, or -10(RNAi) embryos (Fig. 4A) (22, 23). The comparable reductions in CTD phosphorylation found in taf-2(RNAi) embryos suggest that TAF-2 belongs to the same functional class as TAF-1 (Table II).
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The extensive requirements for TAF-1 and TAF-2 for C. elegans transcription are surprising because in S. cerevisiae these TAFIIs have been reported to be essential for transcription of 14 and 3% of the genome, respectively (Table II). In addition, most studies indicate that in yeast the shared TFIID/SAGA TAFIIs Taf-5, Taf-9, and Taf-10 are more broadly required than Taf-1 or Taf-2. In striking contrast, in C. elegans these three shared TFIID/SAGA TAFIIs are needed for a significantly smaller proportion of embryonic transcription than TAF-1 and are dispensable at various metazoan-specific genes that require taf-1 for normal expression levels (Table II). It is possible that some of these differences derive from technical factors. Yeast TAFIIs have been studied in conditional mutant strain populations in the context of ongoing mRNA production, but we depleted TAFIIs before transcription began in individual C. elegans embryos, where maternal mRNAs sustain viability. We believe, however, that these differences may derive from taf-1 and taf-2 having a broader role in C. elegans transcription.
The importance of C. elegans TAF-1 for transcription could derive from its having mechanistic functions that yeast TAF-1 does not. In metazoans but not yeast, TAF-1 contains a C-terminal kinase and a double bromodomain that targets TFIID to acetylated nucleosomes (Fig. 1A) (56). In yeast, related domains are present in the Bdf1 protein, which interacts substoichiometrically with TFIID and in its genetically redundant relative Bdf2 (57). Bdf1 and Bdf2 have metazoan orthologs that are distinct from TAF-1, however, and apparently have TFIID-independent functions in euchromatin maintenance (58, 59). Although it is possible that the TAF-1 bromodomains might have some TFIID-independent functions, the comparable requirement for TAF-2 which we have observed (Fig. 4D) argues against this view.
The simplest model to explain our findings is that a higher percentage of promoters require TFIID in the C. elegans embryo than in yeast. In yeast it seems that a TAFII-independent TBP form is sufficient at many genes and that TFIID occupancy is proportional to transcription only where the TATA element is weak or nonexistent (12, 13). TFIID is recruited to most yeast promoters at low levels, however (7), consistent with it possibly being required more broadly for TBP recruitment in other organisms. Although relatively little is known about C. elegans promoters, fewer than half of surveyed human and Drosophila core promoters contain a TATA element (60), suggesting that TFIID-dependent promoters may be abundant in metazoans. This requirement may not extend to all core promoter elements, however; TAF-6 and TAF-9 bind the downstream promoter element (DPE), a core promoter motif identified in humans and Drosophila, predicting that recognition of a DPE-like element might not be necessary at the many C. elegans genes that do not require TAF-9 for expression (Table II).
In addition to the possibility that promoter recognition by TAF-1 and TAF-2 may be more important in metazoans than in yeast, the relationship among TFIID, activators, and other transcription regulatory factors may be more complex in metazoans. For example, in Drosophila the versatility of combinatorial gene regulation is enhanced by pairing of compatible enhancers and core promoters (60). An interesting aspect of the C. elegans embryo is the importance of the TBP-like protein TLF for transcription (24, 25). Although TLF can direct transcription to TFIID-independent promoters (26), in the C. elegans embryo various genes require both TLF and TAF-1 (see "Results") and possibly TFIID. Some of these genes are conserved in unicellular eukaryotes (Fig. 6), which lack TLF. Our findings suggest that in certain contexts TLF has specialized regulatory functions and acts in concert with TFIID, a model that is consistent with evidence that TLF may influence chromatin organization (26, 61).
It is intriguing that the metazoan-specific histone acetyltransferase CBP-1 apparently has functions that overlap with and complement those of TAF-1 in vivo. Thus, although cbp-1 inhibition eliminated some taf-1-independent gene expression, end-1 is largely taf-1-independent but requires cbp-1 to overcome repression (Fig. 7, A and C) (44). The functional overlap between TAF-1 and CBP-1 could involve their respective histone acetyltransferase functions but could also derive from these proteins contributing to PIC stabilization. It is striking that CBP-1 was required for only a very limited proportion of transcription (Figs. 4C and 7, B and C), given its importance for many differentiation pathways (49). Certain genes, like end-1, may require CBP-1 because their regulation involves particular signals or repressor activities (Fig. 7A).
The broad role in C. elegans embryonic transcription played by TAF-1 and TAF-2 suggests that these TAFIIs and TFIID may generally be of greater importance for transcription in metazoans than predicted from yeast studies. Although TFIID and other PIC components are recruited to promoters in vivo with precise timing, the order of these events varies among different genes, presumably so that their regulation can be tailored to fit particular situations (62). Our findings support the idea that the functional relationships among PIC components and coactivators also vary among species and biological contexts. Elucidating these differences is likely to be important for understanding regulation of metazoan transcription, particularly for unraveling the complexities of tissue- and stage-specific gene regulation.
| FOOTNOTES |
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¶ To whom correspondence should be addressed: Section of Developmental and Stem Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02115. Tel.: 617-919-2769; Fax: 617-730-0023; E-mail: keith.blackwell{at}joslin.harvard.edu.
1 The abbreviations used are: PIC, preinitiation complex; CBP, cAMP-responsive element-binding protein-binding protein; CTD, C-terminal domain; dsRNA, double strand RNA; GFP, green fluorescent protein; Inr, initiator; pol II, RNA polymerase II; RNAi, RNA interference; RT, reverse transcription; SAGA, SPT-ADA-GCN5; TAFII, TATA-binding protein-associated factor; TBP, TATA-binding protein; TF, transcription factor; WT, wild type. ![]()
2 A. Walker, P. Dufourcq, and F. Gay, unpublished observations. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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