Fig. 2. Expression of TAF-5, TAF-10 and TAF-11 in wild-type and RNAi embryos. Representative wild-type or TAF_II 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).

Fig. 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.

Fig. 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%) expressed END-1::GFP in E2 cells (not shown). Lineage analysis (n >5) revealed that all END-1::GFP-positive cells derived from the E cell. Asterisks mark the E4 cells in the ama-1(RNAi) and taf-5(RNAi) embryos. In each of these RNAi embryo sets, E2 descendants were mislocalized to the posterior edge of the embryo because their abnormally short cell cycle resulted in defective gastrulation.

Fig. 1. Similarities between C elegans TAF_IIs and their human and yeast counterparts. (A) C.elegans TAF-5 is compared with hTAF_II130 and yTAF_II48. 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 hTAF_II130. The metazoan-specific conserved elements are important for activator binding by hTAF_II130 (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 TAF_II is indicated below or within the relevant motifs. (B) TAF-10 is related to hTAF_II31/32 and yTAF_II17 within the histone fold (dark gray) (Burley and Roeder, 1996) and an adjacent conserved region (light gray). (C) TAF-11, hTAF_II30 and yTAF_II25 are most similar within the histone fold region (in gray) (Gangloff et al., 2001b).

Fig. 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) TAF_II 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 taf_II(RNAi) embryos. Lineage analysis of each set of taf_II(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.

Fig. 5. Comparable requirements for taf-5, taf-10 and taf-11 at conserved genes. GFP fluorescence was examined in wild-type or taf_II(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.