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Originally published In Press as doi:10.1074/jbc.M310731200 on January 15, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15339-15347, April 9, 2004
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An Extensive Requirement for Transcription Factor IID-specific TAF-1 in Caenorhabditis elegans Embryonic Transcription*

Amy K. Walker{ddagger}§, Yang Shi§, and T. Keith Blackwell{ddagger}§

From the {ddagger}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
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The general transcription factor TFIID sets the mRNA start site and consists of TATA-binding protein and associated factors (TAFIIs), some of which are also present in SPT-ADA-GCN5 (SAGA)-related complexes. In yeast, results of multiple studies indicate that TFIID-specific TAFIIs are not required for the transcription of most genes, implying that intact TFIID may have a surprisingly specialized role in transcription. Relatively little is known about how TAFIIs contribute to metazoan transcription in vivo, especially at developmental and tissue-specific genes. Previously, we investigated functions of four shared TFIID/SAGA TAFIIs in Caenorhabditis elegans. Whereas TAF-4 was required for essentially all embryonic transcription, TAF-5, TAF-9, and TAF-10 were dispensable at multiple developmental and other metazoan-specific promoters. Here we show evidence that in C. elegans embryos transcription of most genes requires TFIID-specific TAF-1. TAF-1 is not as universally required as TAF-4, but it is essential for a greater proportion of transcription than TAF-5, -9, or -10 and is important for transcription of many developmental and other metazoan-specific genes. TAF-2, which binds core promoters with TAF-1, appears to be required for a similarly substantial proportion of transcription. C. elegans TAF-1 overlaps functionally with the coactivator p300/CBP (CBP-1), and at some genes it is required along with the TBP-like protein TLF(TRF2). We conclude that during C. elegans embryogenesis TAF-1 and TFIID have broad roles in transcription and development and that TFIID and TLF may act together at certain promoters. Our findings imply that in metazoans TFIID may be of widespread importance for transcription and for expression of tissue-specific genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic mRNA transcription involves formation of a preinitiation complex (PIC)1 at the core promoter, which directs initiation. The PIC includes a set of general transcription factors (TFIIA, B, D, E, F, and H) and a mediator complex, along with RNA polymerase II (pol II) (1, 2). In Saccharomyces cerevisiae many PIC components have surprisingly specific roles at particular gene subsets (3, 4). Much less is known about how individual PIC components contribute to transcription regulation in metazoans, which have evolved a greater complexity of stage- and tissue-specific gene control and additional genes that are not present in yeast.

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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
C. elegans and Bioinformatics—C. elegans strains were provided to us and maintained as described previously (22). The wild type (WT) strain was N2. TAFIIs are named according to Tora (27), a nomenclature different from that described previously for C. elegans TAFIIs (22). C. elegans taf-1 and taf-2 each reidentified their corresponding human and Drosophila counterparts in GenBank data bases. Alignments were produced by Megalign (DNAStar).

Immunostaining and Fluorescence Analysis—Rabbit 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 {alpha}-TAF-9, {alpha}-TAF-10, {alpha}-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. {alpha}-TBP-1 and {alpha}-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 Analysis—For 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-PCR—N2 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% {beta}-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 Analysis—Control, 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 {alpha}-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 {alpha}-pol II, and goat anti-rabbit (Jackson Immunologicals) for {alpha}-CBP-1. Blots were visualized by enhanced chemiluminescence (Amersham Biosciences).



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  		FIG. 5.
Reduced pol II CTD phosphorylation in taf-1(RNAi) embryo populations. Immunoblots of protein extracts prepared from control (C) or taf-1(RNAi) (T) embryos were probed with anti-phospho-Ser-2, anti-phospho-Ser-5 (H14), and {alpha}-pol II antibodies (see "Experimental Procedures"). Hyper- and hypophosphorylated forms of pol II are designated with an open and closed circle, respectively. In this taf-1(RNAi) sample the dramatically decreased CTD phosphorylation levels that were present are representative, but levels of a loading control (CBP-1) were higher than WT (not shown), suggesting that the seemingly elevated level of total pol II (right panel) is unlikely to be significant.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
taf-1 Is Essential during Early Embryonic Development—To investigate TFIID functions in the early C. elegans embryo, we inhibited TAF-1 expression by RNAi. C. elegans TAF-1 is significantly related to hTAF-1 throughout its length, including predicted functional domains (Fig. 1A). A specific antiserum detected TAF-1 in all WT embryonic nuclei, in oocytes, and in the adult germ line, indicating that taf-1 is maternally expressed (Fig. 2 and data not shown). Accordingly, taf-1 mRNA levels were only modestly reduced when zygotic transcription was prevented by RNAi knock-down of ama-1, the pol II large subunit (Fig. 3D). In taf-1(RNAi) embryos, nuclear TAF-1 antibody staining was eliminated (Fig. 2), and taf-1 mRNA levels were reduced dramatically (Fig. 3D), indicating that TAF-1 expression was decreased significantly. In contrast, levels of multiple other TAFIIs, TBP, and AMA-1 were similar to WT in taf-1(RNAi) embryos (Fig. 2 and data not shown).



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  		FIG. 1.
Predicted C. elegans TAF-1 and TAF-2 proteins. A, C. elegans (Ce) TAF-1 (amino acids numbered above) compared with its human (Hs) and yeast (Sc) counterparts. CeTAF-1 includes predicted N-terminal and C-terminal kinase domains (NT Kinase, CT kinase, respectively), histone acetyltransferase (HAT) and ubiquitin conjugation (Ub) domains, and a bromodomain pair. The bromodomains and CT-kinase have been found only in metazoan TAF-1 orthologs (57). Percentages indicate similarity to human and yeast TAF-1, respectively. B, CeTAF-2 compared with HsTAF-2 as in A. CeTAF-2 is similarly related to S. cerevisiae Taf-2 throughout its length (not shown).

 



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  		FIG. 2.
Expression of TAFIIs, TBP, and TLF in taf-1(RNAi) embryos. Representative WT or taf-1(RNAi) embryos (designated above the columns) were stained with antibodies as indicated, along with DAPI to visualize DNA. Embryos measure ~50 µm.

 



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  		FIG. 3.
Terminal and early cell division phenotypes of taf-1(RNAi) embryos. A, taf-1(RNAi) phenotype. Representative terminally arrested RNAi embryos were examined by differential interference microscopy and compared with a WT embryo that was about to hatch. ama-1(RNAi) and taf-1(RNAi) embryos each ceased development with 90-100 cells (n = 4). ama-1 encodes the pol II large subunit. B, PIE-1::GFP expression in WT and RNAi embryos, examined by fluorescence microscopy. In taf-1(RNAi) embryos, each aspect of PIE-1::GFP germ line and subcellular localization was indistinguishable from WT, including the presence of PIE-1 in germ line RNA-protein P granules (32). C, shortened E2 cell cycle in taf-1(RNAi) embryos. Lineage analysis of taf-1(RNAi) embryos revealed that their early cell division planes were normal except that the E2 cells (Ea and Ep) divided prematurely. Only the EMS cell lineage is shown. D, depletion of taf-1 mRNA in taf-1(RNAi) embryos. RT-PCR was performed with RNA from control (C), ama-1(RNAi) (A), or taf-1(RNAi) (T) embryos. The moderate decrease in taf-1 mRNA in ama-1(RNAi) embryos is consistent with inhibition of zygotic but not maternal taf-1 expression, but in taf-1(RNAi) embryos both maternal and zygotic expression of taf-1 was largely prevented. In contrast, expression of the strictly maternally expressed gene rsp-5 (39, 40) was unaffected by knock-down of either taf-1 or ama-1.

 
C. elegans embryonic development is initially sustained by maternally provided gene products (30). Interference with maternal and zygotic expression of other TAFIIs or PIC components such as ama-1 and ttb-1 (TFIIB) arrests embryonic development at about 100 cells without signs of differentiation, a phenotype that is characteristic of a broad zygotic transcription defect (21-23, 31). taf-1(RNAi) embryos arrested development at a similar stage, apparently without differentiation (Fig. 3A). To evaluate maternal RNA storage we monitored early cell divisions and performed parallel experiments in a transgenic strain that expressed a fusion of the maternally derived germ line protein PIE-1 to GFP. Appropriate localization of PIE-1::GFP depends on at least 20 maternal genes (32). In taf-1(RNAi) embryos PIE-1::GFP expression and localization patterns were normal at every stage (Fig. 3B; not shown), suggesting that storage of maternal gene products was likely to be intact. Early cell division timing and cleavage planes were also normal in these RNAi embryos, except that the cell cycle period of the two E daughters (E2 cells), which give rise to the endoderm, was decreased by approximately half (Fig. 3C). The last phenotype is characteristic of a broad transcription defect (21, 22). Our findings suggest that depletion of embryonic TAF-1 does not detectably alter maternal mRNA stores but may significantly impair embryonic mRNA transcription.

Severely Reduced pol II CTD Phosphorylation Levels in taf-1(RNAi) Embryos—We 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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  		FIG. 4.
Reduced pol II CTD phosphorylation in individual taf-1(RNAi) embryos. A, decreased CTD Ser-2 and Ser-5 phosphorylation in taf-1(RNAi) embryos. Prior to developmental arrest, WT or RNAi embryos (in rows) were stained with anti-phospho-Ser-2 or anti-phospho-Ser-5 antibodies and DAPI to visualize DNA. Representative embryos of comparable stages are presented. In parallel experiments, staining with an antibody against a different RNA pol II region revealed that pol II levels were equivalent in WT and TAFII RNAi embryos (Fig. 2). The relative differences in anti-phospho-Ser-5 and anti-phospho-Ser-2 staining intensities between WT and RNAi embryos were comparable between the onset of transcription at the four-cell stage and terminal arrest and when embryos were photographed at multiple exposure times. Germ line nuclei that are in the focal planes shown are marked with an white asterisk. B, expanded anti-phospho-Ser-5-stained somatic nuclei. In taf-1(RNAi) somatic nuclei, nucleoplasmic anti-phospho-Ser-5 staining is dramatically reduced, but two bright foci are present as in the WT germ line. C, CTD Ser-2 phosphorylation in taf-1(RNAi) and cbp-1(RNAi) embryos, analyzed as in A. Yellow asterisks indicate cells in early stages of mitotic chromosome condensation, in which anti-phospho-Ser-2 stains a pol II-independent cross-reactive epitope (37). D, CTD phosphorylation in taf-2(RNAi) embryos. In this figure, {alpha}PSer5 refers to staining with the P-CTD (A and B) and H14 (D) antibodies, which stain with highly similar patterns (not shown).

 
In the C. elegans embryo, the levels and patterns of anti-phospho-Ser-5 and anti-phospho-Ser-2 staining parallel overall transcription activity (22, 23, 31, 37, 38). Nuclear staining with these antibodies first appears at the three- to four-cell stage, when new mRNA transcription begins. CTD Ser-2 phosphorylation is detected only in the transcriptionally active somatic cells (Fig. 4, A, C, and D) (37). CTD Ser-5 phosphorylation is apparent as a partially punctate nucleoplasmic pattern in interphase somatic nuclei but is limited to two discrete foci in the transcriptionally silent germ line nucleus (Fig. 4, A, B, and D). These germ line foci depend upon the general transcription factor TFIIB and the mediator component RGR-1, suggesting that they require PIC formation (22, 31).

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 ({alpha}-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) Embryos—To 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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  		FIG. 6.
taf-1- and tlf-1-dependent expression of individual genes. A, reduced expression of generally conserved eukaryotic genes in taf-1 and tlf-1(RNAi) embryos. Differential interference (DIC) and fluorescent (FL) images are shown of WT or RNAi embryos (in rows) from the indicated reporter strains. In a representative experiment the nonintegrated RPS-5::GFP reporter was expressed in 18 of 40 WT embryos and 23 of 41 cbp-1(RNAi) embryos, but in none of >50 of each set of taf-1(RNAi) or tlf-1(RNAi) embryos. Embryos shown are otherwise representative of the entire population (>40 embryos) analyzed in each of multiple independent experiments. B, reduced expression of endogenous genes in ama-1 and taf-1(RNAi) embryos. RT-PCR was performed with total RNA from control (C), ama-1(RNAi) (A), or taf-1(RNAi) (T) embryos. Genes shown on the top row are conserved all among eukaryotes, and those on the bottom row are metazoan-specific. In C. elegans the presence of embryonic rsp-5 (solid underline) mRNA does not depend upon embryonic AMA-1 or TAF-1 because this gene is transcribed specifically in the maternal germ line (39, 40). elt-5 and pal-1 (dotted underline) may be expressed both maternally and zygotically (39, 40, 63, 64). Each primer set was tested against at least two independent RNA preparations in multiple experiments. To assure linearity of the reactions, primer sets were used with multiple cDNA dilutions and at different amplification cycle numbers. rsp-5 was tested in each independent experiment to control for the amount of RNA present.

 


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  		TABLE I
Requirements for taf-1 in metazoan-specific gene expression

Expression of the indicated reporters is designated as - for no fluorescence or the background levels seen in ama-1(RNAi) (pol II) embryos, +++ for wild type levels, ++ for intermediate levels of expression, and + for very low levels. Results from ama-1(RNAi) and taf-9(RNAi) experiments are from Ref. 22. For each data set, more than 40 embryos were analyzed in multiple independent experiments.

 
We also analyzed expression of GFP reporters for tissue-specific genes involved in development of the mesentoderm (med-1 and -2), endoderm (end-1), pharynx (pha-4), and epidermis (elt-5) in taf-1(RNAi) embryos. As embryonic transcription begins med-1 and -2 are induced by maternally provided SKN-1 (Fig. 7A) (41), then pha-4 and elt-5 are expressed slightly later (42, 43). end-1 regulation appears to be complex (Fig. 7A), involving MED proteins and the actions of CBP-1 and WNT signaling, which together relieve repression mediated by histone deacetylase (HDA-1) and POP-1 (29, 41, 44). In taf-5, taf-9, and taf-10(RNAi) embryos these genes were expressed at WT levels (Table I) (22, 23). In contrast, in taf-1(RNAi) embryos the med-1, med-2, pha-4, and elt-5 reporters were expressed in normal patterns but at significantly reduced levels (Fig. 7, B and C, and Table I). This residual MED-1::GFP expression was also seen in taf-1, taf-10(RNAi) embryos (Fig. 7B), indicating that it is independent of taf-10. END-1::GFP was expressed at WT levels in ~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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  		FIG. 7.
Defects in expression of developmental genes in taf-1(RNAi) embryos. A, pathways controlling early mesendodermal gene expression in C. elegans. Regulation of end-1 depends on multiple maternal and zygotic inputs (see "Results"). B, expression of MED-1::GFP in RNAi embryos, analyzed as in Fig. 5. C, developmental gene expression in taf-1 and cbp-1(RNAi) embryos. END-1::GFP is expressed specifically in the E cell (endodermal) lineage. In a representative experiment, END-1::GFP was expressed at the E4 and E8 stages at normal levels in (70%; n = 68) of taf-1(RNAi) embryos. end-1 is unique among genes that we have analyzed in that END-1::GFP expression is not necessarily uniform within sets of TAFII RNAi embryos (22, 23). In each RNAi embryo set, E2 descendants were mislocalized to the posterior edge of the embryo because of defective gastrulation (Fig. 3). In all mixed dsRNA experiments a uniform total RNA concentration was maintained using unrelated dsRNA.

 
RT-PCR experiments demonstrated that expression of many endogenous metazoan-specific genes was reduced similarly in taf-1(RNAi) embryos. Expression of the zygotically expressed genes pha-4 and elt-2 was undetectable in ama-1(RNAi) embryos and greatly reduced in taf-1(RNAi) embryos (Fig. 6B). Expression of genes that appear to be expressed both maternally and zygotically expressed (sur-5, elt-5, and pal-1) was reduced but not eliminated in both ama-1 and taf-1(RNAi) embryos (Fig. 6B). Significantly, mRNA production from the corresponding endogenous genes paralleled expression of the PHA-4, ELT-5, and SUR-5::GFP reporters in these RNAi embryos (Figs. 6B and 7C and Table I), suggesting that in general the decreases in reporter expression which we observed in living embryos reflected comparably reduced endogenous e transcription.

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 Genes—In 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) Embryos—The 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have obtained evidence that taf-1 and taf-2 are each required for most mRNA transcription in the C. elegans embryo. In taf-1(RNAi) and taf-2(RNAi) embryos, at every stage nucleoplasmic anti-phospho-Ser-2 and anti-phospho-Ser-5 antibody staining was decreased to levels only slightly higher than background (Fig. 4 and data not shown), indicating that continuous broad reductions in pol II transcription had occurred. Immunoblots of embryonic extracts also showed a striking decrease in pol II CTD phosphorylation (Fig. 5). TFIID-specific TAF-1 was also necessary for normal expression of each metazoan-specific gene that we analyzed, with the exception of end-1 (Figs. 6B and 7 and Table I). This requirement for TAF-1 is much more extensive than revealed by previous analyses performed in metazoans, in which TAF-1 function was not completely ablated (18, 19).

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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  		TABLE II
Requirements for yeast and C. elegans TAF11s for transcription in vivo

In vivo analyses of TAF11 function in C. elegans and S. cerevisiae are summarized.

 
It is intriguing that in each TAFII RNAi embryo set we have analyzed, somatic nuclei contain two discrete anti-phospho-Ser-5 staining foci (Fig. 4B) (22, 23). Similar foci are normally present in the embryonic germ line, where transcription is blocked by PIE-1 (51), a global repressor that appears to act at a postinitiation step (52, 53). These anti-phospho-Ser-5 foci depend upon the presence of the general transcription factor TFIIB, the mediator component RGR-1, and the CTD Ser-5 kinase CDK-7 (22, 38, 54), but not upon the mRNA capping enzyme or the elongation kinase P-TEFb, which are required specifically for Ser-2 phosphorylation (31, 55). The dependence of these anti-phospho-Ser-5 foci on initiation factors suggests that they might derive from aborted or incomplete transcription events and that the lack of TAFIIs blocks some transcription after PIC formation and CTD Ser-5 phosphorylation have occurred.

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
 
* This work was supported by grants from the National Institutes of Health (to T. K. B. and Y. S.) and the March of Dimes (to T. K. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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. Back

2 A. Walker, P. Dufourcq, and F. Gay, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Andrew Fire for constructs; Grace Gill, Steve Buratowski, Johnathan Whetstine, and the Blackwell laboratory members for helpful discussions or critical reading of this manuscript; and Leslie Tu for technical assistance.



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