From the Center for Blood Research and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 29, 2002
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ABSTRACT |
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As conserved components of the transcription
factor (TF) IID- and TFTC/SAGA-related complexes, TATA-binding
protein-associated factors (TAFIIs) are important for
eukaryotic mRNA transcription. In yeast, genetic analyses suggest
that, although some individual TAFIIs are required for
transcription of most genes, others have highly specialized functions.
Much less is known about the functions of TAFIIs in
metazoans, which have more complex genomes that include many
tissue-specific genes. TAF-5 (human (h) TAFII100) is
of particular interest because it is predicted to have an important
structural role. Here we describe the first genetics-based analysis of
TAF-5 in a metazoan. By performing RNA interference in
Caenorhabditis elegans embryos, which can survive for
several cell generations without transcription, we found that
taf-5 is important for a significant fraction of
transcription. However, TAF-5 is apparently not essential for the
expression of multiple developmental and other metazoan-specific genes.
This phenotype remarkably resembles the previously described effects of
similarly depleting two C. elegans histone fold
TAFIIs, TAF-9 (hTAFII31/32) and TAF-10
(hTAFII30), but is distinct from the widespread
transcription block caused by TAF-4 (hTAFII130) depletion.
Our findings suggest that TAF-5, TAF-9, and TAF-10 are part of a
functional module of TFIID- and TFTC/SAGA-related complexes that can be
bypassed in many metazoan-specific genes.
Eukaryotic mRNA transcription requires the coordinate activity
of gene-specific activators, coactivator proteins, general transcription factors
(TFIIA,1 TFIIB, TFIID, TFIIE,
TFIIF, and TFIIH), Mediator complexes, and RNA polymerase II (pol II)
(1-3). This complexity allows the transcription machinery to
communicate with gene-specific regulators through an extraordinary
diversity of combinatorial interactions. Genetic studies performed in
yeast indicate that, although many transcription machinery components
are essential, others seem to perform more specialized roles in
regulating subgroups of genes (4-6). In general, genes involved in
maintenance of cell viability are shared by all eukaryotes, suggesting
that aspects of their regulation are likely to be conserved between
yeast and metazoans. However, most metazoan genes, including those
controlling development and differentiation, are not conserved in
single cell eukaryotes and may require alternative regulatory
strategies (7, 8).
The general transcription factor TFIID is composed of the TATA-binding
protein along with 12-14 additional polypeptides, the TATA-binding
protein-associated factors
(TAFIIs)2
(5, 9, 10). The TAFIIs are generally conserved in
eukaryotes (11). TFIID has various functions during initiation; it
appears to possess enzymatic activities, and TAFIIs have
been implicated in essential interactions with gene-specific activators
and with core promoter sequences located near the transcription start
site (5, 9, 12, 13). Many TAFIIs contain a domain that is related to the histone fold, through which they form dimers within a
conserved TFIID structure (14-17). Some TAFIIs are also
constituents of complexes that lack TATA-binding protein but share some
functions with TFIID, including SAGA and the related metazoan complexes TFTC and PCAF (18-21). TFTC can substitute for TFIID during
transcription initiation (20), and in vivo studies suggest
that, in yeast, the TFIID and SAGA complexes function redundantly in
many genes (6).
In yeast, individual TAFIIs are required for cell
viability, but studies involving conditional mutations or shutoff
systems indicate that TAFIIs differ significantly in the
extent to which they are required for transcription. A consensus has
emerged from these studies that a significant fraction of yeast genes
can be transcribed independently of TAFIIs, that
TAFII dependence maps to core promoters, and that
TAFIIs that are present in both the TFIID and SAGA
complexes are more broadly required than those present in TFIID alone
(5, 6, 9, 12, 13, 22, 23). These models remain a subject of
investigation and debate, however (24, 25).
The striking conservation of TFIID structure predicts that
TAFII functions are likely to be conserved between yeast
and humans. It is an open question, however, how TAFIIs
contribute to regulation of developmental and other metazoan-specific
genes. Investigation of metazoan TAFII functions in
vivo is difficult not only because of cell lethality, but also
because TAFIIs are generally expressed maternally in the
embryo, making their mutant phenotypes complex (26, 27). To circumvent
these issues, we have used RNA interference (RNAi) (28) to inhibit both
maternal and zygotic expression of TAFIIs in the
Caenorhabditis elegans embryo. In the early embryo, maternally produced mRNAs maintain viability until the 100-cell stage in the absence of transcription, making it feasible to manipulate expression of even essential transcription factors (29, 30). We have
previously studied three histone fold TAFIIs: TAF-4, TAF-9, and TAF-10 (31). Surprisingly, TAF-4 appears to be required for
essentially all early embryonic transcription. In contrast, TAF-9 and
TAF-10 are required for a significant fraction of transcription, but
not for expression of many metazoan-specific genes, implying the
existence of a functional TAFII subgroup that is not
essential at many metazoan promoters.
Given the difference between these two classes of C. elegans
TAFII phenotypes, it is of interest to study TAF-5
(human TAFII100), which may mediate critical
interactions within the TFIID and SAGA complexes. TAF-5 contains WD-40
repeats, which generally form a protein-protein interaction module, and
it binds to TAF-9 and multiple other TAFIIs (32-35).
Expression shutoff experiments and analysis of certain conditional
taf5 mutations suggest that, in yeast, Taf5 is necessary for
a small fraction of pol II transcription, but that other
taf5 mutations have more severe effects (6, 36, 37). A
particular taf5 mutation that broadly inhibits transcription
destabilizes most TFIID components and impairs SAGA function,
consistent with Taf5 having important functions in these complexes
(37). Metazoan TAF-5 orthologs have not been studied in
vivo, but a Drosophila TAF-5-related protein
(Cannonball) is expressed specifically in primary spermatocytes and is
required for expression of spermatid differentiation genes (38). The importance of Cannonball for this particular developmental
transcription program raises the question of whether TAF-5 similarly
may be critical for developmental gene regulation.
In this study, we have investigated requirements for taf-5
in the early C. elegans embryo. We found that TAF-5 is
broadly required for transcription, but is remarkably similar
functionally to TAF-9 and TAF-10. In contrast to TAF-4, TAF-5 does not
appear to be required for the expression of multiple metazoan-specific promoters or for a significant fraction of early transcription. In
addition, RNAi co-inhibition experiments suggest that simultaneous lack
of taf-5 and taf-10 does not cause a broader
transcription defect. We conclude that TAF-5, TAF-9, and TAF-10 form
part of a functional module of TFIID- and TFTC/SAGA-related complexes that is not required at many developmental and other metazoan-specific genes.
C. elegans and Bioinformatics--
C. elegans strains
were maintained as described (31). The wild-type strain was N2. Green
fluorescent protein (GFP) reporter strains were provided to us as cited
previously (31). C. elegans taf-5 was identified by
searching WORMpep and genomic data bases (Sanger Center) with human and
Saccharomyces cerevisiae protein sequences. Alignments were
produced by Megalign (DNASTAR, Inc.). The taf-5 open reading
frame is F30F8.8.
Immunostaining and Fluorescence Analysis--
Rabbit antisera
that were raised against the N-terminal TAF-5 peptide
THNNSAMEDNLLSRPMNNES with an N-terminal Cys added were affinity-purified (31). For TAF-5 staining, embryos were subjected to
2% paraformaldehyde fixation and freeze-cracked before treating with
methanol. Washes and antibody incubations were performed in 1×
phosphate-buffered saline, 1% Triton X-100, and 1% bovine serum
albumin prior to staining. Anti-TAF-5 antibody staining was competed by
the cognate (but not heterologous) peptides (data not shown). Staining
with other antibodies, including anti-TAF-9, anti-TAF-10, anti-pol II
(POL 3/3) (39), anti-phospho-Ser-5 (P-CTD) (40), and
anti-phospho-Ser-2 (H5) (Babco), was performed as described (31). For
GFP analysis, embryos were transferred to 2% agarose pads. Images were
captured with a Zeiss AxioSKOP2 microscope and AxioCam digital camera,
and GFP or antibody staining intensities were compared over a range of
exposure times. Pixel intensities were standardized using Adobe
Photoshop Version 5.0.
RNAi Analysis--
A taf-5 cDNA (yk348c7) that
covers >90% of the predicted coding region was obtained from Yuji
Kohara (National Institute of Genetics, Mishima, Japan).
In vitro synthesized double-stranded RNA (Ribomax, Promega)
was injected at 0.6-1.0 µg/µl into young adults (two to eight
fertilized embryos). Uniform populations of terminally arrested embryos
appeared 18-22 h later, and evidence of maternal gene expression
defects (rounded embryos, equal cell division planes) did not appear
until 48 h. For GFP analysis or immunostaining, embryos were
collected from dissected hermaphrodites 24 h after injection.
Embryos were generally obtained from worm pools, but for
END-1::GFP progeny, individual worms were scored. Because
most analyses were performed before terminal arrest, RNAi effectiveness
was confirmed by monitoring sibling embryos that were allowed to
develop. Simultaneous taf-5 and taf-10 RNAi was performed with a 1:1 mixture of double-stranded RNAs. In parallel, a
1:1 dilution of each individual double-stranded RNA with either TE or an unrelated double-stranded RNA (glp-1)
resulted in appropriate terminal arrest, END-1::GFP
expression, and CTD epitope staining levels (data not shown). For heat
shock, hsp-16.2::gfp embryos were
incubated at 37 °C for 1 min in 10 µl of M9 medium.
Fluorescence was examined 1 h later.
taf-5 Is Essential during Early Embryonic Development--
Data
base searches of the C. elegans genome revealed a single,
well conserved taf-5 ortholog (Fig.
1), which re-identified the corresponding
human (TAFII100) and yeast
(TAFII90) genes as its closest
relatives in the GenBankTM/EBI Data Bank. The predicted
TAF-5 protein contains six WD-40 motifs as well as three additional
domains that are conserved in yeast, human, and C. elegans
(Fig. 1). Although yeast Taf5 is present in both TFIID and SAGA
(41), human PCAF complexes contain a related protein, PAF-65
To evaluate the distribution of TAF-5 in early C. elegans
embryos, we examined its expression by antibody staining. TAF-5 was
present in all embryonic nuclei (Fig. 2).
We also noted that TAF-5 was present in oocytes and the adult germ line
(data not shown), suggesting that it is maternally expressed.
Inhibition of taf-5 expression by RNAi eliminated embryonic
staining with the anti-TAF-5 antibody (Fig. 2), suggesting that a
significant depletion of the TAF-5 protein occurred. In contrast,
levels of TAF-4 and TAF-10 did not appear to be affected in
taf-5(RNAi) embryos (Fig. 2). Similarly, antibody staining
indicated that TAF-5 levels were approximately normal in
taf-4(RNAi), taf-9(RNAi), and
taf-10(RNAi) embryos (data not shown).
Maternally deposited RNAs control early developmental patterns and
sustain the C. elegans embryo during early embryogenesis (42). When maternal expression and zygotic expression of essential general transcription factors, including ama-1 (pol
II large subunit), ttb-1 (TFIIB), taf-4,
taf-9, taf-10, cdk-9, and
rgr-1, are inhibited by RNAi, embryonic development arrests
at ~100 cells without differentiation (29, 31, 43, 44). The
development of taf-5(RNAi) embryos arrested at a similar
stage without signs of differentiation (Fig. 3A), suggesting a broad defect
in zygotic transcription.
To investigate whether the taf-5(RNAi) phenotype involves a
general defect in maternal mRNA stores, we evaluated early cell division patterns and performed parallel experiments in a transgenic strain that expresses a fusion of the maternally derived germ line
protein PIE-1 and GFP. PIE-1::GFP recapitulates the
endogenous PIE-1 localization pattern, which depends upon at least 20 maternal genes (45). As in ama-1(RNAi) embryos, in
taf-5(RNAi) embryos, PIE-1::GFP expression and
localization patterns were normal at every stage (Fig. 3B
and data not shown). Early cell division timings and cleavage planes
were also generally normal in taf-5(RNAi) embryos, except
for the cell cycle period of the two E daughters (E2 cells), which give
rise to the endoderm. When early mRNA transcription is broadly
inhibited, as in ama-1(RNAi) embryos, the E2 cell cycle length is shortened from 45 to ~22 min (29, 31). The E2 cells similarly divided after 22 min in taf-5(RNAi) embryos.
Together, our findings suggest that depletion of embryonic TAF-5 does
not detectably influence maternal mRNA stores, but may
significantly impair new mRNA transcription.
Reduced pol II CTD Phosphorylation in taf-5(RNAi)
Embryos--
To investigate how mRNA transcription is
affected in taf-5(RNAi) embryos, we analyzed phosphorylation
of the pol II large subunit CTD. The CTD consists of multiple repeats
that are based on the consensus YSPTSPS (46). Pol II is recruited to
promoters with the CTD in an unphosphorylated form; then during
transcription, the CTD is first phosphorylated at Ser-5 of the repeat
by the TFIIH kinase (40, 47). During elongation, the distribution of
CTD phosphorylation shifts to Ser-2 (47, 48), which, in metazoans, is phosphorylated by the positive-transcription
elongation factor b (P-TEFb) kinase (43, 49). CTD Ser-5 and Ser-2
phosphorylation can be specifically detected in C. elegans
embryonic nuclei by staining with antibodies P-CTD and H5, respectively
(30, 31, 40), which we refer to as anti-phospho-Ser-5 and
anti-phospho-Ser-2 for clarity (Fig.
4).
In the C. elegans embryo, the presence of nuclear
anti-phospho-Ser-5 and anti-phospho-Ser-2 antibody staining is
dependent upon transcription. Staining with these antibodies is not
detectable until the 3-4-cell stage, when new mRNA transcription
begins; then at later stages, the patterns and intensity of this
staining appear to parallel transcription activity (30, 31). CTD Ser-5 phosphorylation was detected as a punctate pattern in the
transcriptionally active somatic nuclei, but was limited to two bright
foci in the transcriptionally silent germ line nucleus (Fig. 4,
A and B). These germ line anti-phospho-Ser-5 foci
depend upon the essential initiation factor TFIIB (TTB-1) and the
Mediator component RGR-1, suggesting that they may correspond to
aborted or stalled transcription events (31, 44). Phosphorylation of
CTD Ser-2 was detected only in somatic cells (Fig. 4A). Both
anti-phospho-Ser-5 and anti-phospho-Ser-2 antibody staining levels are
reduced to background levels when transcription initiation is inhibited
in ttb-1(RNAi) or rgr-1(RNAi) embryos (31,
44).
In taf-5(RNAi) embryos, nucleoplasmic anti-phospho-Ser-5 and
anti-phospho-Ser-2 antibody staining levels were significantly reduced
in parallel, and two anti-phospho-Ser-5 foci like those present in the
germ line were also prominent in somatic nuclei (Fig. 4, A
and B). This pattern suggests a partial but significant reduction in overall embryonic CTD phosphorylation and mRNA
transcription levels, and it is strikingly similar to the pattern seen
in taf-9(RNAi) or taf-10(RNAi) embryos (Fig.
4A) (31). These decreases in staining are distinct, however,
from the more dramatic effects observed in somatic cells in
taf-4(RNAi) embryos, in which anti-phospho-Ser-5 antibody
staining was reduced to only the two foci, and anti-phospho-Ser-2 staining was undetectable (31). Previously, we observed that the
effects of inhibiting expression of taf-9 and
taf-10 simultaneously by RNAi were not distinguishable from
the effects of inhibiting either gene individually (31). Significantly,
anti-phospho-Ser-5 and anti-phospho-Ser-2 antibody staining levels
similarly did not decrease further when taf-5 and
taf-10 were inhibited simultaneously by RNAi
(taf-5,taf-10(RNAi) embryos) (Fig. 4), suggesting that taf-5, taf-9, and taf-10 may be
required for transcription of highly overlapping sets of genes.
Expression of Many Metazoan-specific Genes in taf-5(RNAi)
Embryos--
To evaluate the importance of TAF-5 for expression of
individual genes, we performed RNAi experiments in a set of C. elegans strains that carry transgenic reporters. These reporters
include intact regulatory regions fused to GFP coding regions and are expressed in the embryo in parallel to the corresponding endogenous genes. Expression of each of these reporters is undetectable or reduced
to similar trace levels in ama-1(RNAi) and
taf-4(RNAi) embryos (31).
We first investigated the expression of two groups of genes that are
widely expressed in the C. elegans embryo. rps-5,
let-858, and the heat shock gene hsp-16.2 each
have orthologs in unicellular eukaryotes as well as in metazoans. In
yeast, expression of rps-5 and many other ribosomal protein
genes is highly dependent on TAFIIs (12, 13). Expression of
GFP reporters that correspond to these three conserved genes was
abolished in taf-5(RNAi) embryos (Fig.
5A and Table
I), consistent with a significant
reduction in overall transcription levels (Fig. 4). In contrast, TAF-5
did not appear to be essential in some widely expressed
metazoan-specific genes. cki-2 (cyclin-dependent
kinase inhibitor) and sur-5 (MAPK pathway) are
conserved in metazoans and are expressed early in the C. elegans embryo. The corresponding GFP reporters were expressed at
wild-type levels in taf-5(RNAi) embryos (Table I).
pes-10, which has been identified only in C. elegans, is activated at the onset of embryonic transcription.
PES-10::GFP expression was reduced significantly (but not
eliminated) in taf-5(RNAi) embryos (Table I). Significantly,
each of these various genes was expressed at levels comparable to those
that are characteristic of taf-9(RNAi) or
taf-10(RNAi) embryos (31).
We also analyzed expression of a group of developmental genes in
taf-5(RNAi) embryos. These genes specify or promote
differentiation of the mesendoderm (med-1 and
med-2), endoderm (end-1), pharynx (pha-4), and epidermis (elt-5). GFP reporters
that correspond to med-1, med-2, and
elt-5 were expressed at wild-type levels in all
taf-5(RNAi) embryos, and END-1::GFP was similarly
expressed in ~30% of these RNAi embryos (Fig. 5B and
Table I). PHA-4::GFP was also robustly expressed in
taf-5(RNAi) embryos, but in fewer cells than in wild-type
embryos (Table I). Significantly, in each case, these expression
patterns closely paralleled those observed previously in
taf-9(RNAi) and taf-10(RNAi) embryos (31), with
the exception that END-1::GFP was expressed in a higher
proportion (80-90%) in taf-9(RNAi) and
taf-10(RNAi) embryos. In addition, both MED-1::GFP
and END-1::GFP were expressed comparably in
taf-5(RNAi) and taf-5,taf-10(RNAi) embryos (Fig.
5B). Together, our findings indicate that TAF-5 is essential
for a significant proportion of early embryonic transcription, but not
for expression of many metazoan-specific genes, a phenotype that is
remarkably similar to the previously described requirements for TAF-9
and TAF-10.
Much remains to be learned about how general transcription
machinery components participate in regulating different types of genes
in metazoans. In this study, we have obtained evidence that, in the
early C. elegans embryo, TAF-5 (human
TAFII100) is required for a significant fraction of pol II
transcription, but does not appear to be essential for expression of
many metazoan-specific genes. Overall levels of pol II CTD Ser-5 and
Ser-2 phosphorylation were substantially reduced (but not eliminated)
throughout development of taf-5(RNAi) embryos (Fig. 4,
A and B), implying a significant but incomplete
defect in pol II transcription. Accordingly, in these RNAi embryos, a
set of conserved genes was not expressed detectably, but various
developmental and other metazoan-specific genes were transcribed at
significant levels (Fig. 5 and Table I).
Multiple lines of evidence argue against the notion that this limited
requirement for TAF-5 might have derived from incomplete RNAi
penetrance, although we cannot eliminate the possibility that trace
levels of the TAF-5 protein may remain in these RNAi embryos. These
RNAi effects were accompanied by loss of anti-TAF-5 antibody staining
and were highly reproducible, and they appeared with consistent timing
after injection (Fig. 2 and data not shown). In addition, expression of
the conserved genes let-858, rps-5, and
hsp-16.2 was not detected in taf-5(RNAi) embryos
(Fig. 5A). Finally, the taf-5(RNAi) phenotype did
not appear to be enhanced by simultaneous RNAi inhibition of
taf-10 (Figs. 4A and 5B), suggesting that these respective RNAi phenotypes involve overlapping processes and
are unlikely to be partial effects.
In S. cerevisiae, expression shutoff analyses and various
conditional alleles suggest that Taf5 has a limited role in
transcription, but other taf5 alleles are associated with
more severe defects that correlate with functional or structural
impairment of TFIID and SAGA (36, 37). A particular yeast
taf5 mutation that causes a very broad transcription defect
is also associated with destabilization of most other TFIID subunits
(37). We cannot currently address whether the TFIID complex is intact
in our experiments. In taf-5(RNAi) embryos, approximately
normal levels of TAF-10 and the broadly essential TAF-4 appeared to be
present, however (Fig. 2), suggesting that the taf-5(RNAi)
phenotype does not involve a general loss of TFIID or TFTC/SAGA subunits.
A particularly interesting aspect of our findings is the remarkable
similarity between the taf-5(RNAi) phenotype and the
previously described effects of inhibiting expression of
taf-9 or taf-10, either alone or simultaneously
(31). The evidence that taf-5(RNAi) and
taf-5,taf-10(RNAi) embryos are phenotypically similar (Figs. 4 (A and B) and 5B) further supports
the model that TAF-5 and TAF-10 are functionally linked. Our evidence
that many early embryonic genes are expressed independently of TAF-5,
TAF-9, and TAF-10 appears to be consistent with genetic and promoter
occupancy studies suggesting that a significant proportion of S. cerevisiae genes are transcribed independently of all
TAFIIs (13, 22, 23). We have previously observed,
however, that essentially all early embryonic transcription appears to
require taf-4 (31), indicating that this yeast model may not
be fully applicable to C. elegans.
It is striking that TAF-5, TAF-9, and TAF-10 do not seem to be required
to express nearly all of the developmental and other metazoan-specific
genes that we have analyzed (Fig. 5B and Table I) (31). We
conclude that these three TAFIIs are part of a functional
subgroup that can be bypassed during transcription of many
metazoan-specific genes. Although many of the genes involved in basic
cellular functions have been highly conserved between yeast and
metazoans, genes that control processes that are specific to
multicellular animals, such as development and differentiation, are
much more distantly related (7, 8). Our findings suggest that,
although many conserved genes such as rps-5 may have
retained regulatory strategies that require these TAFIIs
(Fig. 5A), many metazoan-specific genes have evolved
alternative activation mechanisms, perhaps involving different core
promoter contexts and activator or coactivator interactions. Because
TAF-5 does not appear to be generally required in embryonic
developmental gene expression programs, the importance of
Drosophila Cannonball for spermatid differentiation gene
transcription is particularly intriguing (38). Perhaps a specialized
form of TFIID or TFTC/SAGA that contains Cannonball has evolved to
perform a highly specialized developmental regulatory function. Future
elucidation of the differences and parallels between TAF-5 and
Cannonball functions may therefore reveal novel mechanistic insights
into how TAFIIs contribute to transcription.
The apparent parallels between TAF-5, TAF-9, and TAF-10 functions
suggest that these TAFIIs may be located along a shared surface for protein-protein or protein-DNA interactions within TFIID
and TFTC/SAGA complexes. It is consistent with this model that TAF-5
interacts with TAF-9 and with multiple other TAFIIs (32,
34, 35). The differences between the apparently general requirement for
TAF-4 and the more limited functions of TAF-5, TAF-9, and TAF-10 raise
the interesting question of what accounts for these differences at the
molecular level. In vivo analyses of additional
TAFIIs, coupled with a more detailed understanding of TFIID
and TFTC complex structure, should reveal mechanisms through which
individual TAFIIs and different domains of TFIID and TFTC
complexes are utilized or bypassed during transcription of metazoan genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(19).
Using the above search criteria, we did not detect a PAF-65
ortholog, suggesting that, in C. elegans, TAF-5 is
utilized in both TFIID- and TFTC/SAGA-related complexes. We also did
not identify a C. elegans ortholog of Drosophila Cannonball.
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Fig. 1.
Domain conservation in C. elegans
TAF-5. C. elegans (Ce) TAF-5
(amino acids numbered above) includes six WD-40 motifs (red)
and three other conserved domains (CD1-CD3;
blue), all of which are present in yeast and other metazoan
TAF-5 proteins (34). Percent similarity to human (h) TAF-5
is shown in each box, whereas percentages indicated below
indicate similarity to human PAF-65 .
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Fig. 2.
Expression of TAF-5, TAF-4, and
TAF-10 in wild-type and taf-5(RNAi) embryos.
Representative wild-type (WT) or taf-5(RNAi)
embryos were stained with antibody (AB) to TAF-5, TAF-4,
TAF-10, or the pol II large subunit AMA-1 (antibody POL 3/3), along
with 4,6-diamidino-2-phenylindole (DAPI) to visualize DNA.
Embryos measure ~50 µm.
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Fig. 3.
Terminal and early cell division phenotypes
of taf-5(RNAi) embryos. A,
taf-5(RNAi) phenotype. Representative terminally arrested
RNAi embryos were examined by differential interference microscopy and
compared with a wild-type (WT) embryo that was about to
hatch. ama-1(RNAi) and taf-5(RNAi) embryos each
ceased development with 90-100 cells (n = 5).
ama-1 encodes the pol II large subunit. B,
PIE-1::GFP expression in wild-type and RNAi embryos, examined
by fluorescence microscopy. In taf-5(RNAi) embryos, each
aspect of the PIE-1::GFP germ line and subcellular
localization was indistinguishable from that in wild-type embryos,
including the presence of PIE-1 in germ line RNA-protein P granules
(45).
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Fig. 4.
Reduced pol II CTD phosphorylation in
taf-5(RNAi) embryos. A, comparably
decreased CTD Ser-5 and Ser-2 phosphorylation in taf-5(RNAi)
and taf-10(RNAi) embryos. Prior to terminal developmental
arrest, wild-type (WT) or RNAi embryos (in rows)
were stained with anti-phospho-Ser-5 ( -PSer5) or
anti-phospho-Ser-2 (
-PSer2) antibody and
4,6-diamidino-2-phenylindole (DAPI) to visualize DNA.
Representative embryos of comparable stages are presented. In parallel
experiments, staining with an antibody against a different pol II
region revealed that pol II levels were equivalent in wild-type and
TAF11(RNAi) embryos (Fig. 2). The relative
differences in anti-phospho-Ser-5 and anti-phospho-Ser-2 antibody
staining intensities between wild-type and RNAi embryos were comparable
between the activation of transcription at the 4-cell stage and
terminal arrest, and when embryos were photographed at multiple
different exposure times. Germ line nuclei that are in the focal plane
shown are marked with white arrows, and somatic nuclei
depicted in B are indicated by a red arrow.
Anti-phospho-Ser-2 antibody variably cross-reacted with germ line
RNA-protein P granules, as in the taf-10(RNAi) embryo.
B, detailed view of anti-phospho-Ser-5 antibody-stained
wild-type somatic (SOM) and germ line (GL)
nuclei, along with ama-1(RNAi) and taf-5(RNAi)
somatic nuclei. In the wild-type germ line nucleus (A,
white arrows), note the presence of two discrete foci and
the lack of nucleoplasmic staining that is evident in somatic nuclei.
In taf-5(RNAi) somatic nuclei, these foci are present along
with significantly reduced nucleoplasmic staining.
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Fig. 5.
Expression of individual conserved and
metazoan-specific genes in taf-5(RNAi) embryos.
A, taf-5-dependent conserved
genes. Differential interference (DIC) and fluorescent
(FL) images are shown of wild-type (WT) and RNAi
embryos (in rows) from the reporter strains indicated. Each
of these reporters was expressed in most embryonic cells. In a
representative experiment, the RPS-5::GFP reporter, which is
nonintegrated, was expressed in 21 of 45 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. B, expression of developmental genes in
taf-5(RNAi) embryos. Expression of MED-1::GFP and
END-1::GFP was examined in wild-type and RNAi embryos.
In a representative experiment, END-1::GFP was expressed at
normal levels (30%; n = 71) in taf-5(RNAi)
embryos. end-1 is unique of the genes that we have analyzed
in that END-1::GFP expression is not necessarily uniform
within sets of TAF11(RNAi) embryos (31); this
may reflect its highly complex regulation, which involves multiple
signal inputs (50, 51). In each RNAi embryo set, E2 descendants were
mislocalized to the posterior edge of the embryo due to defective
gastrulation caused by the reduced E2 cell cycle time (see
"Results").
Requirements for taf-5 for early embryonic gene expression
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Grace Gill, Yang Shi, and Blackwell laboratory members for helpful discussions and critically reading this manuscript and Leslie Tu for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant GM62891 from the National Institutes of Health (to T. K. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for Blood
Research, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3150; Fax: 617-278-3153; E-mail:
blackwell@cbr.med.harvard.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M211056200
2 The TAFII nomenclature follows that of Tora (11) and differs from the C. elegans TAFII names we used in our previous work (31).
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ABBREVIATIONS |
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The abbreviations used are: TF, transcription factor; pol II, RNA polymerase II; TAF, TATA-binding protein-associated factor; RNAi, RNA interference; GFP, green fluorescent protein; CTD, C-terminal domain; MAPK, mitogen-activated protein kinase.
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