Taf3 is Required for Normal Development and Hematopoiesis in Early Zebrafish Embryos
As a first step toward delineating the function of Taf3, we examined the expression profile of the taf3
gene in early zebrafish embryos. The RT-PCR analysis of shows that the taf3
transcript was first detected at 4 hours post fertilization (hpf), coincident with the onset of zygotic transcription. By contrast, the Taf3 protein was detected as early as the one cell stage embryo (0 hpf; ), indicating that the protein is maternally deposited, similar to the Trf3 protein (Hart et al., 2007
). Whole-mount in situ hybridization analysis revealed that taf3
mRNA was widely distributed throughout the zebrafish embryo with no discernible tissue-restriction during the first 24 hours of development ().
Fig. 1 Taf3 is required for normal development and hematopoiesis in early zebrafish embryos. A: RT-PCR analysis monitoring expression of taf3 and, as a control, gapdh in zebrafish embryos at 0, 4, 8 and 24 hpf. B: Immunoblot analysis monitoring expression of (more ...)
To investigate the role of Taf3 in zebrafish development, we used a morpholino oligonucleotide (MO) to ablate Taf3 function. We first performed immunoblot analysis to confirm that injection of the taf3
MO resulted in decreased levels of Taf3 (Supp. Fig. S1
). shows that injection of the taf3
MO into one-cell stage fertilized zebrafish embryos resulted in a retardation of development and gross morphological defects in late somitogenesis. These developmental defects were strikingly similar to those observed in Trf3-depleted embryos (Hart et al., 2007
), and bore a strong resemblance to Cdx4-depleted embryos ( and Davidson et al., 2003
) and the Cdx4 mutant, kugelig
(Hammerschmidt et al., 1996
To determine whether Taf3-depleted embryos, like Trf3- and Cdx4-depleted embryos, failed to undergo hematopoiesis, we analyzed Taf3 morphants for expression of several hematopoietic markers. First, we analyzed expression of gata1
, which is required for the expression of a variety of genes in the erythroid lineage. The whole-mount in situ hybridization experiment of (top panel) shows that the characteristic expression pattern of gata1
in the lateral mesoderm was lost in Taf3-depleted embryos and restored by injection of taf3
mRNA bearing a silent mutation that prevented hybridization with the taf3
MO. Expression of a second lateral mesoderm marker, pax2
, was unaffected by loss of Taf3, consistent with our previous finding that pax2
expression is not dependent upon Trf3 (Hart et al., 2007
). We also examined expression of gata1
using a transgenic line in which the fluorescent protein dsRed is under control of the endogenous gata1
promoter (Traver et al., 2003
). (bottom panel) shows that expression of the gata1:dsRed
transgene was greatly reduced in Taf3-depleted embryos (n=168/255). In addition, we used in situ hybridization to monitor expression of two other hematopoietic markers, scl
, which were both reduced in Taf3-depleted embryos (). Expression of pu.1
, a marker of myeloid cells that arise in the anterior lateral mesoderm, was also reduced in the absence of Taf3 (Supp. Fig. S2
), consistent with our previous findings that pu.1
expression is diminished in Trf3- and Mespa-depleted embryos (Hart et al., 2007
It has been previously shown that some MOs can non-specifically activate a p53 pathway resulting in apparent development defects (Robu et al., 2007
). We therefore performed a series of experiments, the results of which conclusively demonstrate that the developmental and hematopoietic defects we observed in Taf3-depleted embryos were not due to non-specific p53 activation (Supp. Fig. S3
). Collectively, the results of indicate that, like Trf3, Taf3 is required for zebrafish hematopoiesis.
Taf3 is Selectively Recruited to the Promoter of the Trf3 Target Gene mespa in vivo and is Required for mespa Expression
As stated above, we previously identified mespa
as a direct Trf3 target gene; Trf3, but not Tbp, is recruited to the mespa
promoter and is required for mespa
expression (Hart et al., 2007
). The RT-PCR analysis of shows that mespa
mRNA levels were significantly reduced in Taf3-depleted embryos, indicating that Taf3 is also required for mespa
expression. Chromatin immunoprecipitation (ChIP) experiments in wild-type zebrafish embryos confirmed that Taf3 was bound to the mespa
promoter (). Collectively, these results indicate that Taf3 is an essential component of the pre-initiation complex involved in mespa
Fig. 2 Taf3 is selectively recruited to the promoter of the Trf3 target gene mespa in vivo and is required for mespa expression. A: RT-PCR analysis monitoring expression of mespa in control and taf3 MO-treated embryos at 6 hpf. B: ChIP analysis monitoring occupancy (more ...)
We have previously shown that the developmental and hematopoietic defects of Trf3-depleted embryos can be rescued by ectopic expression of mespa
(Hart et al., 2007
). We therefore examined whether mespa
expression could similarly rescue the defects of Taf3-depleted embryos. shows that injection of mespa
mRNA restored normal expression of gata1
MO-injected embryos, consistent with our conclusion that mespa
is a downstream target of Taf3.
Taf3 Selectively Interacts with the C-terminal Domain of Trf3 but not Tbp
We next investigated whether, as in mouse cell lines (Deato and Tjian, 2007
), zebrafish Trf3 and Taf3 physically interact. Plasmids expressing Flag-tagged Taf3 and either myc-tagged Trf3 or Tbp were co-transfected into COS-7 cells. After 48 hours, extracts were prepared, Taf3 was immunoprecipitated with an anti-Flag antibody, and the immunoprecipitates were analyzed for the presence of Trf3 or Tbp by immunoblotting with an anti-myc antibody. The results of show that Taf3 interacted with Trf3 but not Tbp.
Fig. 3 Taf3 selectively interacts with the C-terminal domain of Trf3 but not Tbp. A: Co-immunoprecipitation assays. Plasmids expressing Flag-tagged Taf3 and either myc-tagged Trf3, Tbp or Trf3/Tbp chimeric protein were co-transfected into COS-7 cells. Taf3 was (more ...)
As stated above, TRF3 has a C-terminal DNA-binding domain nearly identical to that of TBP, whereas the N-terminal domain is highly divergent. It therefore seemed likely that residues in the N-terminus would provide specificity for Taf3 association, and that a Trf3 derivative lacking the N-terminus would be unable to interact with Taf3. To test this hypothesis, we performed a domain-swapping experiment. We constructed a set of myc-tagged chimeric proteins consisting of the N-terminus of Trf3 fused to the C-terminus of Tbp [Trf3(N)-Tbp(C)] or the N-terminus of Tbp fused to the C-terminus of Trf3 [Tbp(N)-Trf3(C)], and tested the ability of these chimeric proteins to co-immunoprecipitate with Flag-tagged Taf3. Unexpectedly, we found that the Tbp(N)-Trf3(C) but not the Trf3(N)-Tbp(C) chimeric protein interacted with Taf3 (), suggesting that the determinants required for interaction with Taf3 reside within the C-terminus of Trf3.
As a control, we next performed a similar set of experiments with TAF1, which directly interacts with TBP and is believed to function as a scaffold of the TFIID complex (Chen et al., 1994
). Because the zebrafish Taf1 had not yet been cloned, we used human TAF1, which is 76% identical to the zebrafish protein. Co-immunoprecipitation analysis showed that TAF1 interacted with zebrafish Tbp but not Trf3 (). Domain-swapping experiments using the chimeric proteins described above revealed that the interaction between Tbp and TAF1 was mediated by the C-terminus of Tbp. Collectively the results of suggest that residues within the C-terminal conserved domains of Tbp and Trf3 are responsible for selective interaction with Taf1 and Taf3, respectively.
To confirm this conclusion we attempted to isolate Trf3 mutants that were defective for interaction with Taf3. Of the 192 amino acids in the C-terminal domain of Trf3, only 14 differ from the corresponding residue in the C-terminal domain of Tbp (Supp. Fig. S4
). Within these 14 amino acids we generated a series of site-directed double amino acid substitutions that changed Trf3 residues to the corresponding Tbp sequence (). This approach enabled us to identify a pair of amino acid substitutions, E291V and S293G (hereafter called site-directed mutant 1 or SDM1), which abolished the ability of Trf3 to interact with Taf3 (). Notably, these two amino acids also differ between the human TRF3 and TBP proteins (Persengiev et al., 2003
Interaction with Taf3 is Required for Trf3 Function in Zebrafish Development
We used the Trf3(SDM1) mutant to ask whether the Trf3-Taf3 interaction was required for Trf3 function in vivo. Synthetic mRNAs encoding Trf3, Tbp, Trf3(SDM1) and as a control another Trf3 mutant harboring a pair of amino acid substitutions, A149G and P151K (hereafter referred to as SDM2), which retains interaction with Taf3 (see ), were injected into one cell stage Trf3 morphants and analyzed for their ability to rescue morphological development. As expected, injection of a trf3 mRNA bearing a silent mutation that prevented hybridization with the trf3 MO restored normal morphological development whereas injection of a tbp mRNA did not (). Injection of an mRNA encoding the Trf3(SDM2) mutant was also able to rescue normal development. By contrast, injection of an mRNA encoding the Trf3(SDM1) mutant failed to rescue development.
Fig. 4 Interaction with Taf3 is required for Trf3 function in zebrafish development. A: Whole-mount in situ hybridization with a riboprobe to hbae1 (22 hpf) in trf3 MO-treated embryos injected with mRNAs expressing tbp, trf3 or a trf3 mutant. Arrowheads denote (more ...)
To assess the completeness of rescue in greater detail, we analyzed expression of several hematopoietic markers. The in situ hybridization results of show that expression of hbae1, a terminal marker of erythroid cell fate, was rescued by injection of an mRNA encoding the Trf3(SDM2) mutant but not the Trf3(SDM1) mutant. Similar results were obtained for all other hematopoietic markers analyzed, including gata1 () and earlier markers of hematopoietic precursors, scl1 and lmo2 (). These results indicate that interaction with Taf3 is required for Trf3 function in zebrafish development and hematopoiesis.
TRF3 and TAF3 are also Required for Initiation of Hematopoiesis in the Mouse
The pathways required for hematopoietic development are in general highly conserved throughout vertebrates, although some notable differences have been documented (reviewed in Fossett and Schulz, 2001
). We therefore asked whether TRF3 and TAF3 had roles in initiation of hematopoiesis in the mouse similar to those in zebrafish embryos. We first analyzed whether, as in zebrafish (Hart et al., 2007
), there was a TRF3-MESP1-CDX4 transcription factor pathway in mouse (MESP1 is the murine orthologue of zebrafish Mespa). In the initial experiments we tested whether mouse Mesp1
was, like zebrafish mespa
, a Trf3 target gene. shows, identical to the results obtained in zebrafish embryos, that in embryonic day 10.5 (E10.5) mouse embryos TRF3 but not TBP was bound selectively to the Mesp1
Fig. 5 Requirement of TRF3 and TAF3 for expression of mouse Mesp1. A: ChIP analysis monitoring occupancy of TRF3, TBP, RNA polymerase II (POL2) or, as a negative control (C), an irrelevant protein (yeast Gal4) on the Mesp1 and Actin promoters in mouse E10.5 (more ...)
We also performed ChIP analysis in mouse F9 embryonic teratocarcinoma cells, a well-established model system for investigating early events in mouse development (Strickland and Mahdavi, 1978
; Fischer et al., 2000
; Kubota et al., 2001
). shows that in F9 cells the Mesp1
promoter was also selectively bound by TRF3 but not TBP. Significantly, the Mesp1
promoter in F9 cells was also bound by TAF3. When F9 cells were differentiated by treatment with retinoic acid, Trf3
expression was rapidly extinguished followed by loss of Mesp1
expression (), raising the possibility that in F9 cells TRF3 was required for Mesp1
To confirm this conclusion we performed an RNA interference (RNAi) experiment. shows that following transfection of Trf3 double-stranded RNA (dsRNA) into F9 cells, Trf3 expression levels were, as expected, substantially reduced. By contrast, expression of Tbp and Gapdh were unaffected, confirming the specificity of RNAi-mediated TRF3 depletion. Significantly, expression of Mesp1 was also dramatically reduced, indicating that TRF3 is required for Mesp1 expression. RNAi-mediated depletion of TAF3 also resulted in loss of Mesp1 expression (). Thus, analogous to the results in zebrafish embryos, Mesp1 is a TRF3 target gene in mouse embryos and F9 teratocarcinoma cells. In addition, as in zebrafish embryos, in F9 teratocarcinoma cells TAF3 is an essential TRF3 co-factor.
Mouse embryonic stem (ES) cells, when differentiated as suspension aggregates called embryoid bodies, give rise to differentiated hematopoietic cells (reviewed in Choi et al., 2005
). We analyzed the expression profile of Trf3
in mouse embryoid bodies. The RT-PCR results of show that Trf3
were first detected at very early times following differentiation (day 1). Trf3
expression was substantially decreased by day 3 followed by the loss of Mesp1
expression by day 4. As expected, Taf3
, which is also a component of the GTF TFIID, was present at a relatively constant level throughout the 6-day time course (Supp. Fig. S5
Fig. 6 Requirement of TRF3 for initiation of hematopoiesis in mouse embryoid bodies. A: RT-PCR analysis monitoring expression of various genes in mouse embryoid bodies at the indicated times (days) following induction of differentiation. B: Mouse embryonic stem (more ...)
Definitive markers of hematopoiesis, such as Lmo2 and Scl, first appeared on day 3, and Gata1 was first detected on day 3 and subsequently increased markedly. By contrast, expression of mesoderm/endothelial markers, such as Brachyury and Kdr, first appeared on day 0, prior to expression of Trf3, Mesp1 or Cdx4. Thus, the temporal expression pattern in mouse embryoid bodies also supports the involvement of a TRF3-MESP1-CDX4 pathway in hematopoiesis.
Finally, to provide functional evidence for the role of TRF3 in hematopoietic development in mouse embryoid bodies we performed an RNAi experiment. A small interfering RNA (siRNA) was used to knockdown TRF3 in mouse ES cells (Supp. Fig. S6
), followed by differentiation and analysis of hematopoietic markers. The results of show that TRF3 knockdown resulted in markedly decreased levels of Scl
, indicative of decreased initiation of hematopoiesis. Thus, as in zebrafish embryos, TRF3 is required to initiate hematopoiesis in mouse embryoid bodies.