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An Extensive Requirement for Transcription Factor IID-specific TAF-1 in Caenorhabditis elegans Embryonic Transcription

2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês

10.1074/jbc.m310731200

ISSN

1083-351X

Autores

Amy K. Walker, Yang Shi, T. Keith Blackwell,

Tópico(s)

Plant Molecular Biology Research

Resumo

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. 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. Eukaryotic mRNA transcription involves formation of a preinitiation complex (PIC) 1The 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. 1The 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. 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) (1Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 2Hochheimer A. Tjian R. Genes Dev. 2003; 17: 1309-1320Crossref PubMed Scopus (194) Google Scholar). In Saccharomyces cerevisiae many PIC components have surprisingly specific roles at particular gene subsets (3Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1594) Google Scholar, 4Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (299) Google Scholar). 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 (5Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (271) Google Scholar, 6Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). 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 (5Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (271) Google Scholar, 6Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 7Mencia M. Moqtaderi Z. Geisberg J.V. Kuras L. Struhl K. Mol. Cell. 2002; 9: 823-833Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 8Li X.-Y. Bhaumik S.R. Zhu X. Li L. Shen W.-C. Dixit B.L. Green M.R. Curr. Biol. 2002; 12: 1240-1244Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). TAF-1 and TAF-2 together bind directly to the initiator (Inr) element, which encompasses the start site (9Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (176) Google Scholar). TAF-1, the largest TAFII, is also necessary for TFIID stability and possesses histone acetyltransferase, kinase, and ubiquitin conjugating activities (10Wassarman D.A. Sauer F. J. Cell Sci. 2001; 114: 2895-2902Crossref PubMed Google Scholar). TAF-1 is unique to TFIID, but many other TAFIIs are also found in the SPT-ADA-GCN5 (SAGA)-related complexes (5Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (271) Google Scholar, 6Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), 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 (5Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (271) Google Scholar, 6Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). 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 (3Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1594) Google Scholar, 4Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (299) Google Scholar, 11Shen W.C. Bhaumik S.R. Causton H.C. Simon I. Zhu X. Jennings E.G. Wang T.H. Young R.A. Green M.R. EMBO J. 2003; 22: 3395-3402Crossref PubMed Scopus (80) Google Scholar), and chromatin immunoprecipitation has detected significant TAFII occupancy only at TFIID-dependent genes (12Kuras L. Kosa P. Mencia M. Struhl K. Science. 2000; 288: 1244-1248Crossref PubMed Scopus (145) Google Scholar, 13Li X.Y. Bhaumik S.R. Green M.R. Science. 2000; 288: 1242-1244Crossref PubMed Scopus (156) Google Scholar). 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 (14Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (52) Google Scholar). 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 (4Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (299) Google Scholar, 5Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (271) Google Scholar, 6Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). 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 (15Andel F. Ladurner A.G. Inouye C. Tjian R. Nogales E. Science. 1999; 286: 2153-2156Crossref PubMed Scopus (114) Google Scholar, 16Brand M. Leurent C. Mallouh V. Tora L. Schultz P. Science. 1999; 286: 2151-2153Crossref PubMed Scopus (101) Google Scholar, 17Leurent C. Sanders S. Ruhlmann C. Mallouh V. Weil P.A. Kirschner D.B. Tora L. Schultz P. EMBO J. 2002; 21: 3424-3433Crossref PubMed Scopus (78) Google Scholar), 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 (2Hochheimer A. Tjian R. Genes Dev. 2003; 17: 1309-1320Crossref PubMed Scopus (194) Google Scholar). 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 (18Wassarman D.A. Aoyagi N. Pile L.A. Schlag E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1154-1159Crossref PubMed Scopus (41) Google Scholar). In hamster cells a conditional taf-1 mutation decreased expression of ∼18% of genes and caused apoptosis (19O'Brien T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2456-2461Crossref PubMed Scopus (53) Google Scholar), 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) (20Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11654) Google Scholar) 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 (21Powell-Coffman J.A. Knight J. Wood W.B. Dev. Biol. 1996; 178: 472-483Crossref PubMed Scopus (99) Google Scholar). Using this strategy, we determined previously that TAF-4 is required for essentially all early embryonic transcription (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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 (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar, 23Walker A.K. Blackwell T.K. J. Biol. Chem. 2003; 278: 6181-6186Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). 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 (24Dantonel J.C. Quintin S. Lakatos L. Labouesse M. Tora L. Mol. Cell. 2000; 6: 715-722Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 25Kaltenbach L. Horner M.A. Rothman J.H. Mango S.E. Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Hochheimer A. Zhou S. Zheng S. Holmes M.C. Tjian R. Nature. 2002; 420: 439-445Crossref PubMed Scopus (170) Google Scholar). 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. C. elegans and Bioinformatics—C. elegans strains were provided to us and maintained as described previously (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). The wild type (WT) strain was N2. TAFIIs are named according to Tora (27Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar), a nomenclature different from that described previously for C. elegans TAFIIs (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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 (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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 (1× 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 α-TAF-9, α-TAF-10, α-pol II (pol 3/3) (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar), P-CTD (anti-phospho-Ser-5) (28Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (299) Google Scholar), H5 (anti-phospho-Ser-2) (Covance), and CBP-1 (29Victor M. Bei Y. Gay F. Calvo D. Mello C. Shi Y. EMBO Rep. 2002; 3: 50-55Crossref PubMed Scopus (25) Google Scholar) was performed as in Ref. 22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar. α-TBP-1 and α-TLF-1 immunostaining was performed as in Ref. 25Kaltenbach L. Horner M.A. Rothman J.H. Mango S.E. Mol. Cell. 2000; 6: 705-713Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar. 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. (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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. (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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% β-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 α-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 α-pol II, and goat anti-rabbit (Jackson Immunologicals) for α-CBP-1. Blots were visualized by enhanced chemiluminescence (Amersham Biosciences). 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).Fig. 2Expression 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Terminal 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 (32Reese K.J. Dunn M.A. Waddle J.A. Seydoux G. Mol. Cell. 2000; 6: 445-455Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). 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 (39Hill A. Hunter C.P. Tsung B. Tucker-Kellogg G. Brown E. Science. 2000; 290: 809-812Crossref PubMed Scopus (245) Google Scholar, 40Baugh L. Hill A. Slonim D. Brown E. Hunter C. Development. 2003; 130: 889-900Crossref PubMed Scopus (211) Google Scholar) was unaffected by knock-down of either taf-1 or ama-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) C. elegans embryonic development is initially sustained by maternally provided gene products (30Newman-Smith E.D. Rothman J.H. Curr. Opin. Genet. Dev. 1998; 8: 472-480Crossref PubMed Scopus (30) Google Scholar). 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 (21Powell-Coffman J.A. Knight J. Wood W.B. Dev. Biol. 1996; 178: 472-483Crossref PubMed Scopus (99) Google Scholar, 22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar, 23Walker A.K. Blackwell T.K. J. Biol. Chem. 2003; 278: 6181-6186Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 31Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (164) Google Scholar). 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 (32Reese K.J. Dunn M.A. Waddle J.A. Seydoux G. Mol. Cell. 2000; 6: 445-455Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). 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 (21Powell-Coffman J.A. Knight J. Wood W.B. Dev. Biol. 1996; 178: 472-483Crossref PubMed Scopus (99) Google Scholar, 22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar). 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 (33Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). 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 (28Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (299) Google Scholar, 34Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (795) Google Scholar). During elongation the distribution of CTD phosphorylation shifts to Ser-2 (34Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (795) Google Scholar, 35Cho E.J. Kobor M.S. Kim M. Greenblatt J. Buratowski S. Genes Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (338) Google Scholar), which is phosphorylated by the P-TEFb kinase (31Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (164) Google Scholar, 36Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (566) Google Scholar). 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 (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar, 28Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (299) Google Scholar, 37Seydoux G. Dunn M.A. Development. 1997; 124: 2191-2201PubMed Google Scholar), which we refer to as anti-phospho-Ser-5 and anti-phospho-Ser-2 for clarity (Figs. 4 and 5). In the C. elegans embryo, the levels and patterns of anti-phospho-Ser-5 and anti-phospho-Ser-2 staining parallel overall transcription activity (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar, 23Walker A.K. Blackwell T.K. J. Biol. Chem. 2003; 278: 6181-6186Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 31Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (164) Google Scholar, 37Seydoux G. Dunn M.A. Development. 1997; 124: 2191-2201PubMed Google Scholar, 38Shim E.Y. Walker A.K. Blackwell T.K. J. Biol. Chem. 2002; 277: 30413-30416Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). 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) (37Seydoux G. Dunn M.A. Development. 1997; 124: 2191-2201PubMed Google Scholar). 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 (22Walker A.K. Rothman J.H. Shi Y. Blackwell T.K. EMBO J. 2001; 20: 5269-5279Crossref PubMed Scopus (42) Google Scholar, 31Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (164) Google Scholar). In taf-1(RNAi) embryos nucle

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