Distinct requirements for C.elegans TAFIIs in early embryonic transcription
2001; Springer Nature; Volume: 20; Issue: 18 Linguagem: Inglês
10.1093/emboj/20.18.5269
ISSN1460-2075
AutoresAmy K. Walker, Joel H. Rothman, Yang Shi, T. Keith Blackwell,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle17 September 2001free access Distinct requirements for C.elegans TAFIIs in early embryonic transcription Amy K. Walker Amy K. Walker Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Joel H. Rothman Joel H. Rothman Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, CA, 93106 USA Search for more papers by this author Yang Shi Yang Shi Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author T.Keith Blackwell Corresponding Author T.Keith Blackwell Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Amy K. Walker Amy K. Walker Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Joel H. Rothman Joel H. Rothman Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, CA, 93106 USA Search for more papers by this author Yang Shi Yang Shi Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author T.Keith Blackwell Corresponding Author T.Keith Blackwell Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Author Information Amy K. Walker1,2, Joel H. Rothman3, Yang Shi2 and T.Keith Blackwell 1,2 1Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA 2Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA, 02115 USA 3Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, CA, 93106 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5269-5279https://doi.org/10.1093/emboj/20.18.5269 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TAFIIs are conserved components of the TFIID, TFTC and SAGA-related mRNA transcription complexes. In yeast (y), yTAFII17 is required broadly for transcription, but various other TAFIIs appear to have more specialized functions. It is important to determine how TAFIIs contribute to transcription in metazoans, which have larger and more diverse genomes. We have examined TAFII functions in early Caenorhabditis elegans embryos, which can survive without transcription for several cell generations. We show that taf-10 (yTAFII17) and taf-11 (yTAFII25) are required for a significant fraction of transcription, but apparently are not needed for expression of multiple developmental and other metazoan-specific genes. In contrast, taf-5 (yTAFII48; human TAFII130) seems to be required for essentially all early embryonic mRNA transcription. We conclude that TAF-10 and TAF-11 have modular functions in metazoans, and can be bypassed at many metazoan-specific genes. The broad involvement of TAF-5 in mRNA transcription in vivo suggests a requirement for either TFIID or a TFTC-like complex. Introduction Eukaryotic mRNA transcription requires assembly of a multiprotein pre-initiation complex (PIC) at promoters. This machinery includes RNA polymerase (Pol II), general transcription factors (GTFs) required for Pol II activity (TFIIA, B, D, E, F and H) and a mediator-related complex (Hampsey, 1998; Lemon and Tjian, 2000; Malik and Roeder, 2000). Some PIC components are essential for transcription, but in yeast others may act as modular interfaces through which gene groups can be regulated coordinately (Holstege et al., 1998; Green, 2000; Lee et al., 2000). In metazoans, additional PIC components and transcription cofactors have evolved that are not present in yeast (Lemon and Tjian, 2000). Most metazoan genes do not appear to correspond directly to yeast genes, even though many encode conserved domains (Chervitz et al., 1998; Rubin et al., 2000; Rubin, 2001). Given these differences, it is important to determine how conserved PIC components contribute to transcription in metazoans. The GTF TFIID, which recognizes the transcription start site, is remarkably conserved from yeast to humans (Burley and Roeder, 1996; Albright and Tjian, 2000; Green, 2000). TFIID consists of the TATA-binding protein (TBP), along with ∼12 polypeptides known as the TAFIIs (TBP-associated factors). Some TAFIIs interact with core promoter sequences, and various individual TAFIIs can bind a diverse array of upstream transactivators. In addition, human (h) TAFII250 and its orthologs have enzymatic activities that include a conserved histone acetyl transferase (HAT) (Albright and Tjian, 2000; Green, 2000; Matangkasombut et al., 2000; Pham and Sauer, 2000). The TAFIIs may thus provide a functional link between proximal and distal promoter regions, and activities that promote transcription. Consistent with this idea, a TFIID structure reveals surfaces that could mediate extensive core promoter and protein—protein contacts (Andel et al., 1999; Brand et al., 1999a). Some TAFIIs are also present in the human TBP-free TAFII-containing complex (TFTC), and in the related complexes PCAF and STAGA (human) and SPT—ADA—GCN5 (SAGA) (yeast) (Martinez et al., 1998; Ogryzko et al., 1998; Wieczorek et al., 1998; Brand et al., 1999b; Sterner and Berger, 2000). We refer to these as TFTC-related complexes. They lack TBP, and contain either the GCN5 or PCAF HAT instead of an hTAFII250 ortholog. In addition to TAFIIs, TFTC-related complexes contain subunits that are related to TFIID-specific TAFIIs, suggesting possible functional overlap. Supporting this idea, TFTC is structurally similar to TFIID, and can mediate transcription initiation in vitro (Wieczorek et al., 1998; Brand et al., 1999a). Analysis of conditional yeast mutants indicates that expression of most genes depends upon either the TFIID or SAGA HAT, and that many yeast genes may be regulated through the action of either complex (Lee et al., 2000). Individual yeast TAFIIs are each necessary for cell viability, but the extent to which they are required for Pol II transcription in vivo remains controversial (Albright and Tjian, 2000; Green, 2000; Kuras et al., 2000; Li et al., 2000). Some yeast TAFIIs are broadly required, but others appear to have more specific functions that derive from interactions with core promoters, and possibly with other proteins. It appears likely that individual metazoan TAFIIs function analogously to yeast TAFIIs in regulating genes that are conserved in all eukaryotes. It is an open question, however, to what extent they are important at genes that do not have yeast counterparts, which we refer to as metazoan-specific genes. Analysis of metazoan TAFII function in vivo has been hampered by cell lethality, and by the complexity of terminal developmental phenotypes (Zhou et al., 1998; Pham et al., 1999; Wassarman et al., 2000). To circumvent the problem of cell lethality, we are studying metazoan TAFIIs in the Caenorhabditis elegans embryo. Caenorhabditis elegans embryonic mRNA transcription appears to begin at the 4-cell stage, but in its absence maternally produced mRNAs maintain viability until around the 100-cell stage (Powell-Coffman et al., 1996; Seydoux and Dunn, 1997). In this context, we can investigate the functions of otherwise essential transcription factors in living cells. We have used RNA-mediated interference (RNAi) to investigate the functions of three C.elegans TAFIIs: TAF-5, TAF-10 and TAF-11 (Figure 1; Table I). TAF-10 is of considerable interest because it is orthologous to yeast (y)TAFII17, which is very broadly essential for Saccharomyces cerevisiae transcription (Apone et al., 1998; Michel et al., 1998; Moqtaderi et al., 1998; Lee et al., 2000) (Figure 1B). TAF-11 corresponds to yTAFII25, which has been proposed to be either universally (Sanders et al., 1999) or narrowly (Lee et al., 2000) required (Figure 1C). TAF-5 corresponds to yTAFII48, the requirements for which are unknown (Sanders and Weil, 2000). TAF-5 is particularly interesting because it corresponds to hTAFII130, which contains metazoan-specific motifs that are targeted directly by numerous activators (Figure 1A) (Saluja et al., 1998; Rojo-Niersbach et al., 1999). In addition to being present in TFIID, TAF-10 and TAF-11 orthologs are found in all TFTC-related complexes, and a TAF-5 ortholog is present in TFTC (see Brand et al., 1999b). We show that C.elegans taf-10 and taf-11 are required for a significant proportion of embryonic transcription but, strikingly, are not limiting for activation of multiple developmental and other metazoan-specific genes. In contrast, taf-5 appears to be essential for virtually all early transcription. Our findings suggest that TAF-10 and TAF-11 form part of a functional module that is not required for activation of many metazoan-specific genes, and that TAF-5 may have a more fundamental mechanistic role. Figure 1.Similarities between C elegans TAFIIs and their human and yeast counterparts. (A) C.elegans TAF-5 is compared with hTAFII130 and yTAFII48. TAF-5 includes conserved regions (CR) I and II (speckled boxes), the predicted histone fold (black box) (Gangloff et al., 2000) and glutamine-rich regions (gray boxes) present in hTAFII130. The metazoan-specific conserved elements are important for activator binding by hTAFII130 (Saluja et al., 1998; Rojo-Niersbach et al., 1999), particularly a small motif indicated by an asterisk. In (A), (B) and (C), the percentage similarity to the corresponding human TAFII is indicated below or within the relevant motifs. (B) TAF-10 is related to hTAFII31/32 and yTAFII17 within the histone fold (dark gray) (Burley and Roeder, 1996) and an adjacent conserved region (light gray). (C) TAF-11, hTAFII30 and yTAFII25 are most similar within the histone fold region (in gray) (Gangloff et al., 2001b). Download figure Download PowerPoint Table 1. Caenorhabditis elegans TAFIIs Human Drosophila Saccharomyces cerevisiae C.elegans clone Predicted mol. wt C.elegans name 250 250/230 130/145 W04A8.7 204.0 taf-1 150 150 150 Y37E11B.4 137.0 taf-2 80/70 60/62 60 W09B6.2 80.9 taf-3.1 80/70 60/62 60 Y37E11AL.8 91.8 taf-3.2 100 80/58 90 F30F8.8 72.1 taf-4 130 110 48 R119.6 60.4 taf-5 18 19 C14A4.10 47.0 taf-6 28 30β 40 F48D6.1 37.8 taf-7.1 28 30β 40 K10D3.3 37.6 taf-7.2 28 30β 40 F43D9.5 24.5 taf-7.3 55 67 F54F7.1 29.2 taf-8.1 55 67 Y111B2A.16 26.4 taf-8.2 20 30α 68/61 Y56A4.3 28.0 taf-9 31/32 40/42 17 T12D8.7 20.6 taf-10 30 24 25 K03B4.3 19.6 taf-11 Caenorhabditis elegans homologs of TAFIIs were identified by searching WORMpep or genomic databases (Sanger Centre) with human, Drosophila or S.cerevisiae sequences. The open reading frame, predicted molecular weight and gene name are listed for the C.elegans homologs. Caenorhabditis elegans TAFII genes have been identified and described independently by Aoyagi and Wassarman (2000). Results Caenorhabditis elegans TAFIIs By searching C.elegans databases, we have identified at least one well-conserved homolog for each TAFII in human TFIID (Table I). We have named these C.elegans TAFIIs in order of their predicted molecular weights. Each contains conserved sequence motifs found in their metazoan and yeast orthologs (not shown), including the histone fold domains through which multiple TAFIIs form heterodimers (Burley and Roeder, 1996; Gangloff et al., 2000, 2001a,b). TAF-5, TAF-10 and TAF-11 each contain a characteristic histone fold (Figure 1), through which they are each predicted to pair with a different TAFII (Burley and Roeder, 1996; Gangloff et al., 2000, 2001b). These similarities predict that TFIID structure and function have been conserved in C.elegans. We have also searched for C.elegans orthologs of other TFTC-related complex components. Caenorhabditis elegans encodes a GCN5/PCAF-related HAT (Y.Shi, unpublished) and a TRA1/TRRAP homolog (not shown) but, by our search criteria (Materials and methods), we did not identify orthologs of ADA/SPT proteins (ADA1, ADA2, ADA3, SPT3, SPT7, SPT8 and SPT20), which are not essential for yeast viability (Sterner and Berger, 2000). In C.elegans, ADA/SPT functions may be fulfilled by more distantly related proteins, or a streamlined version of TFTC may be formed by TAFIIs, the GCN5/PCAF-related HAT and TRA1/TRRAP. tafII(RNAi) embryos arrest development early, without differentiation Caenorhabditis elegans embryonic development is orchestrated initially by maternally derived proteins and mRNAs, which establish early cell asymmetries and patterns of new embryonic transcription (Newman-Smith and Rothman, 1998). To determine whether TAF-5, TAF-10 and TAF-11 proteins are present in the early embryo, we examined their expression by staining with peptide-derived antibodies. Under staining conditions that were optimized for early embryos (Figure 2 and data not shown), TAF-5 was apparent in nuclei from the 2-cell stage through early morphogenesis, and TAF-10 and TAF-11 were readily detectable in nuclei from the 4-cell stage through late gastrulation. TAF-5, TAF-10 and TAF-11 were similarly detectable in adult germline and oocyte nuclei (not shown), suggesting that they are maternally expressed. Figure 2.Expression of TAF-5, TAF-10 and TAF-11 in wild-type and RNAi embryos. Representative wild-type or TAFII RNAi embryos (indicated in rows) were stained with antibodies to TAF-5, TAF-10 or TAF-11, or with DAPI to visualize DNA, as indicated above the columns. RNAi embryos were collected 24 h after injection of hermaphrodite mothers, when a uniformly affected population was being produced (see Materials and methods). Download figure Download PowerPoint To investigate taf-5, taf-10 and taf-11 functions in the early embryo, we inhibited their expression by RNAi (Fire et al., 1998). As a benchmark for phenotypes caused by pleiotropic transcription defects, we compared tafII(RNAi) embryos with ama-1(RNAi) and ttb-1(RNAi) embryos. ama-1 encodes the Pol II large subunit (Powell-Coffman et al., 1996) and ttb-1 encodes TFIIB, a Pol II GTF required for transcription initiation (Lemon and Tjian, 2000). We determined whether maternal gene expression was generally intact in these RNAi embryos by monitoring early cell division patterns and timing, and by performing parallel RNAi experiments in a transgenic strain that expresses a fusion of the maternally derived germline protein PIE-1 to green fluorescent protein (GFP). This PIE-1::GFP protein recapitulates the complex patterns of PIE-1 expression and localization, which depend upon >20 other maternal genes (Tenenhaus et al., 1998; Reese et al., 2000b). All ama-1, ttb-1, taf-5, taf-10 and taf-11(RNAi) embryos arrested development at 90–100 cells and lacked signs of differentiation (Figure 3A), as reported previously for ama-1 (Powell-Coffman et al., 1996). At every stage prior to terminal arrest, maternal PIE-1::GFP expression and localization patterns appeared normal in these RNAi embryos (Figure 3A and data not shown). Their early cell division timing and cleavage planes were also generally normal, except for the cell cycle period of the two E cell daughters (E2 cells), which form the endoderm (Figure 3B and data not shown). For gastrulation to occur, the E2 cells must divide after 45 min instead of the 22 min characteristic of their cousins, the two MS2 cells. This cell cycle lengthening requires new mRNA transcription and endodermal specification (Powell-Coffman et al., 1996; Zhu et al., 1998). In ama-1(RNAi), ttb-1(RNAi) and each set of tafII(RNAi) embryos, the E2 cells divided immediately after the MS2 cells (Figure 3B). Our findings suggest that in tafII(RNAi) embryos, maternal mRNA stores appear generally to be intact, but new mRNA transcription may be severely impaired. Figure 3.Terminal and early cell division phenotypes of ama-1 (RNA pol II), ttb-1 (TFIIB), taf-5, taf-10 and taf-11 RNAi embryos. (A) TAFII RNAi embryo phenotypes. RNAi embryos produced by N2 (wild-type) or pie-1::gfp mothers were examined by differential interference (DIC) or fluorescence (FL) microscopy. Typical examples of wild-type (WT) or RNAi embryos are shown, as indicated to the right of each row. The left column compares terminally arrested RNAi embryos with a wild-type embryo that is about to hatch. ama-1(RNAi), ttb-1(RNAi), taf-5(RNAi), taf-10(RNAi) and taf-11(RNAi) embryos each arrested with 90–100 cells (n = 5).The right two columns show 4-cell pie-1::gfp WT and RNAi embryos. In these RNAi embryos, each aspect of PIE-1::GFP germline and subcellular localization was indistinguishable from wild type, including the presence of PIE-1 in germline RNA—protein P granules (Reese et al., 2000b). Embryos measure ∼50 μm. (B) Shortened E2 cell cycle in tafII(RNAi) embryos. Lineage analysis of each set of tafII(RNAi) embryos (n ≥5) revealed that their early cell division planes and times were normal, except that their E2 cells (Ea and Ep) divided prematurely. Only the EMS cell lineage is shown. Download figure Download PowerPoint Nuclear antibody staining for TAF-5, TAF-10 or TAF-11 was eliminated in each respective set of RNAi embryos (Figure 2), indicating a penetrant loss of function. In yeast, loss of some TAFIIs destabilizes other TFIID components (Apone et al., 1998; Michel et al., 1998; Moqtaderi et al., 1998; Chen and Manley, 2000). To investigate whether this might have occurred, we stained tafII(RNAi) embryos with antibodies against each TAFII that we analyzed (Figure 2). TAF-10 and TAF-11 were both present at normal levels in taf-5(RNAi) embryos. Interference with either taf-10 or taf-11 did not affect TAF-5 expression, but caused loss of both TAF-10 and TAF-11. Because we have not detected evidence of maternal gene expression defects in these tafII(RNAi) embryos, we conclude that TAF-10 and TAF-11 proteins may each depend upon each other for stability. Inhibition of Pol II CTD phosphorylation in tafII(RNAi) embryos To investigate overall transcription levels in tafII(RNAi) embryos, we analyzed phosphorylation of the Pol II large subunit C-terminal domain (CTD). The CTD consensus repeat (YSPTSPS; 42 copies in C.elegans) is phosphorylated on actively transcribing Pol II (Hirose and Manley, 2000). CTD phosphorylation is important for promoter clearance, elongation and integration of transcription with mRNA processing. At the promoter, yeast Pol II is phosphorylated on Ser5 of the CTD repeat by the TFIIH kinase (Komarnitsky et al., 2000; Schroeder et al., 2000). As Pol II moves away from the start site, the distribution of CTD phosphorylation shifts to Ser2, but the kinase responsible has not been identified (Komarnitsky et al., 2000). In C.elegans embryos, CTD phosphorylation patterns are tightly correlated with transcriptional activity (Seydoux and Dunn, 1997; Tenenhaus et al., 1998). In somatic nuclei, antibody staining first detects Ser5 phosphorylation as a bright punctate pattern at the 4-cell stage, when transcription begins (Seydoux and Dunn, 1997) (Figure 4, columns 2 and 3). In the transcriptionally silent early germline precursor nucleus, this staining is confined to two distinct foci. Ser2 phosphorylation is first detectable at the 4-cell stage, and is absent in the early embryonic germline (Seydoux and Dunn, 1997) (Figure 4, column 5). Figure 4.Broader requirement for taf-5 than taf-10 or taf-11 for Pol II CTD phosphorylation. Wild-type or RNAi embryos were stained with Pol II CTD antibodies (see text) prior to terminal developmental arrest. Representative embryos of comparable stages are presented in rows, as indicated. Columns 1, 4 and 6 show nuclei stained by DAPI. α-P-Ser5 staining is shown in column 2, and an expansion of the nucleus marked by the red arrow is shown in column 3. Column 5 shows embryos stained with the α-P-Ser2 antibody. Column 7 shows staining with α-UnP CTD; identical results were obtained with an antibody against a different Pol II region (POL 3/3; not shown). α-PSer5 and α-PSer2 recognize Pol II isoforms associated with transcription initiation and elongation, respectively (Komarnitsky et al., 2000; Schroeder et al., 2000), and the α-UnP CTD antibody provides a Pol II expression control. In columns 2 and 5, germline nuclei that are in the focal plane shown are marked with an asterisk. In α-P-Ser5-stained germline nuclei, note the lack of nucleoplasmic staining and the presence of two discrete dots (Seydoux and Dunn, 1997). Some germline nuclei stained with α-PSer2 have perinuclear background deriving from cross-reactivity of the secondary antibodies with P granules. The relative differences in α-PSer5 and α-PSer2 staining intensities were comparable when embryos were photographed at multiple different exposure times. Download figure Download PowerPoint We stained embryos with the P-CTD antiserum to detect phospho-Ser5 (Schroeder et al., 2000), the H5 antibody to detect phospho-Ser2 (Bregman et al., 1995; Patturajan et al., 1998) and the 8WG16 antibody to detect the unphosphorylated CTD (Patturajan et al., 1998). To avoid confusion, we refer to these antibodies as α-PSer5, α-PSer2 and α-UnP CTD, respectively. In contrast to ama-1(RNAi) embryos, in which staining with α-UnP CTD was abolished, in ttb-1(RNAi) and tafII(RNAi) embryos Pol II levels were not significantly affected (Figure 4, column 7). α-PSer5 staining of wild-type embryos (Figure 4, columns 2 and 3) recapitulated the pattern obtained previously with the phospho-Ser5 antibody H14 (see above) (Seydoux and Dunn, 1997). In contrast, nuclear α-PSer5 staining was reduced to background levels in ama-1(RNAi) and ttb-1(RNAi) embryos. In taf-5(RNAi) embryos, diffuse nucleoplasmic α-PSer5 staining was reduced similarly, but each somatic nucleus contained two distinct foci like those in the transcriptionally silent germline cell (Figure 4, columns 2 and 3). In parallel to staining with α-PSer5, α-PSer2 reactivity was comparably severely reduced in ama-1(RNAi), ttb-1 (RNAi) and taf-5(RNAi) embryos (Figure 4, column 5). These staining reductions were representative of these RNAi embryos from the 4-cell stage until arrest (not shown), suggesting that in ama-1(RNAi), ttb-1(RNAi) and taf-5(RNAi) embryos overall transcription levels are extremely low at each embryonic stage. In contrast, CTD phosphorylation was less severely affected in taf-10(RNAi), taf-11(RNAi) and taf-10(RNAi); taf-11(RNAi) embryos. In the somatic nuclei, at all embryonic stages, two α-PSer5 foci were accompanied by nucleoplasmic staining that was decreased, but more prominent than in taf-5(RNAi) embryos (Figure 4, columns 2 and 3; not shown). In taf-10(RNAi), taf-11(RNAi) and taf-10(RNAi); taf-11(RNAi) embryos, α-PSer2 staining levels were similarly not eliminated (Figure 4, column 5). The comparable reduction in CTD phosphorylation accompanying simultaneous interference with taf-10 and taf-11 is consistent with the interdependence of TAF-10 and TAF-11 protein levels (Figure 2). These findings suggest that some transcription can occur independently of taf-10 and taf-11. taf-10 and taf-11 are not rate limiting for many metazoan-specific promoters To investigate how these TAFIIs are involved in expression of individual genes, we performed RNAi experiments in C.elegans that carry transgenic reporters that are transcribed in the early embryo. These reporters include intact regulatory regions along with GFP-fused coding regions, and are expressed in patterns that parallel the corresponding endogenous genes. We examined expression of three genes that are common to yeast and metazoans: let-858, rps-5, and hsp16.2, a heat shock gene. In yeast, rps-5 transcription is highly dependent upon TAFIIs (Li et al., 2000). Interference with taf-5, taf-10 or taf-11 abolished LET-858::GFP and RPS-5::GFP expression, and significantly decreased expression of HSP-16.2::GFP in response to heat shock (Figure 5). Interference with each of these tafIIs comparably impaired expression of these reporters (Figure 5), suggesting that the less severe decrease in CTD phosphorylation associated with interference with taf-10 or taf-11 (Figure 4) reflects a difference in function, not RNAi penetrance. Figure 5.Comparable requirements for taf-5, taf-10 and taf-11 at conserved genes. GFP fluorescence was examined in wild-type or tafII(RNAi) embryos (in rows) that were produced by the reporter strains indicated above the columns. Each of these reporters was expressed in most embryonic cells. In a representative experiment, the RPS-5::GFP reporter, which is non-integrated, was expressed in 23/47 wild-type embryos but in none of >50 of each set of RNAi embryos. Embryos shown are otherwise representative of the entire population analyzed in each of multiple independent experiments, in which >40 embryos were scored per reporter strain. HSP16.2::GFP expression varied slightly within each set of embryos, but those depicted correspond to average levels of expression and to representative differences between WT and RNAi embryos. Genes that are conserved between yeast and metazoans are indicated at the bottom. Download figure Download PowerPoint We next tested how interference with these TAFII genes affects expression of genes that are not present in yeast, but are widely expressed. pes-10 has been identified only in C.elegans, and is expressed at the onset of embryonic transcription (Seydoux and Fire, 1994). PES-10::GFP expression was severely affected by interference with ama-1 or taf-5 expression, and was decreased in taf-10 (RNAi) or taf-11(RNAi) embryos (Figure 5). cki-2 (CDK inhibitor) and sur-5 (MAP kinase pathway component) are broadly conserved among metazoans (Gu et al., 1998; Hong et al., 1998). cki-2 and sur-5 reporters required ama-1 and taf-5, but were unaffected by interference with either taf-10 or taf-11 expression (Table II). Table 2. Requirements for TAFIIs for metazoan-specific gene expression SUR-5::GFP (MAP kinase pathway) CKI-2::GFP (cell cycle) MED-1::GFP (endoderm/mesoderm) MED-2::GFP (endoderm/mesoderm) PHA-4::GFP (digestive tract) ELT-5::GFP (ectoderm) Wild type + + + + + + ama-1(RNAi) − − − − − − taf-5(RNAi) − − − − − − taf-10(RNAi) + + + + + + taf-11(RNAi) + + + + + + Reporter strains were scored as + when GFP was expressed at wild-type levels in all embryos. They were scored as − when GFP was undetectable, or was present at comparable trace levels in tafII and ama-1 RNAi embryos. In each case, >40 embryos were analyzed in multiple independent experiments. In three independent reporter strains, PHA-4::GFP was expressed at normal levels, but in fewer cells than wild type. We also investigated the importance of these TAFIIs for activation of cell type-specific genes. The redundant GATA factor-encoding genes med-1 and med-2 specify mesendodermal lineages (Maduro et al., 2001), and are required for expression of the related gene end-1, which specifies the endoderm (Zhu et al., 1998). pha-4 is a forkhead-family gene that later specifies the pharynx and rectum (Horner et al., 1998; Kalb et al., 1998), and elt-5 encodes an epidermally expressed GATA factor (J.Rothman, unpublished). In taf-5(RNAi) embryos, GFP reporters corresponding to these genes were not expressed above the trace or undetectable levels characteristic of ama-1(RNAi) embryos (Figure 6; Table II). In contrast, med-1, med-2 and elt-5 reporters were expressed robustly in all taf-10(RNAi) and taf-11(RNAi) embryos (Table II). All taf-10(RNAi) and taf-11(RNAi) embryos also expressed PHA-4::GFP in many cells (Table II), a striking finding because pha-4 transcription requires upstream zygotic gene expression (Horner et al., 1998; Kalb et al., 1998). END-1::GFP normally appears in the E2 cells, then persists in their E4—E8 descendants (Figure 6 and data not shown). As predicted from their shortened E2 cell cycle (Figure 3B), in most taf-10(RNAi), taf-11(RNAi) and taf-10(RNAi); taf-11(RNAi) embryos, END-1::GFP initially appeared at normal levels in E4 cells, then was present in E8 cells (Figure 6). These reporter experiments confirm the general importance of taf-5, and suggest that taf-10 and taf-11 are required for a significant fraction of embryonic transcription, but not for expression of many metazoan-specific genes. Figure 6.taf-5, taf-10 and taf-11 are essential for gastrulation, but vary in importance for END-1::GFP expression. END-1::GFP expression was examined in RNAi embryos, as indicated to the right of each row. Differential interference (DIC) and fluorescent (FL) images of an E4 and an E8 stage embryo from each set are shown. In a representative experiment, END-1::GFP was not expressed in any ama-1(RNAi) or taf-5(RNAi) embryos (n >100), but at the E4 and E8 stages was expressed at normal levels in most taf-10(RNAi) (90%; n = 71) and taf-11(RNAi) (80%; n = 82) embryos. In parallel experiments, END-1::GFP was expressed in a similar proportion of taf-10(RNAi); taf-11(RNAi) embryos. Within these last RNAi embryo sets, only a small proportion (<5%) expr
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