TBP and TAFII135 are selectively depleted in extracts from T-RA differentiated F9 cells
F9 cells are a well characterised model for cellular differentiation. In the presence of T-RA, F9 cells differentiate into primitive or visceral endoderm, and into parietal endoderm in the presence of T-RA and bt2
]]. Differentiation is accompanied by growth inhibition, characteristic changes in cell morphology, targeted degradation of RARγ2, induction of marker genes, and increased apoptosis.
To determine whether components of basal transcription factor TFIID were subject to regulation during F9 cell differentiation, immunoblotting with monoclonal antibodies against TBP and a number of TAFIIs, was used to monitor the levels of the respective proteins in differentiated cell extracts (see Materials and Methods).
No significant changes in the levels of TAFII100, TAFII55, TAFII20, TAFII30, and TAFII18, or the largest subunit of RNA polymerase II were observed between 3 and 10 days of T-RA treatment (Fig. , lanes 1-6 and data not shown). In striking contrast, TAFII135 and TBP were strongly and selectively depleted in extracts made from cells differentiated with T-RA for more than 3 days (Fig. lanes 1-2) and remained at low levels up until at least day 10 (lanes 2-5). No such changes were observed in vehicle treated control cultures (Fig. , lanes 7-12). In these experiments, TAFII135 and TBP were detected using two different monoclonal antibodies directed against distinct epitopes (see Materials and methods). The relevant extracts from T-RA treated cells were also analysed by staining with Coomassie Brilliant Blue showing that there was no overall change in the protein profile (Fig. ). Therefore, there is a selective depletion of these two TFIID components in extracts from differentiating cells.
Figure 1 A. Depletion of TBP and TAFII135 in extracts from differentiated F9 cells. Replica plates of cells were treated with T-RA (or vehicle, right panel) at day 0 and extracts were subsequently prepared form one 10 cm plate at the day indicated above each lane. (more ...)
To evaluate the levels of TBP and TAFII135 in extracts from primitive endodermal cells we performed titration experiments using serial dilutions of undifferentiated F9 cell extract. Upon prolonged exposure, TAFII135 could still be detected in a 20-fold dilution of the F9 cell extract, whereas it was virtually undetectable in extracts from day 7 differentiated cells (Fig. , lanes 1-5 compared with lane 6). Similarly, TBP was readily detected in the 20-fold dilution of F9 cell extract, while the levels detected in the differentiated cell extracts were significantly lower (compare lanes 5 and 6).
The above experiments were performed with total cell extracts made in the presence of 0.5 M KCl. We also asked whether the depletion of TBP and TAFII135 would be observed in nuclear extracts from differentiated F9 cells where the nuclei were extracted with 1.0 M KCl to ensure efficient protein extraction. As observed in total cell extracts, TAFII135 and TBP were selectively depleted in nuclear extracts from differentiated cells (Fig. ).
TBP and TAFII135 were strongly and selectively depleted in extracts from visceral endoderm differentiated F9 cells analogous to what was observed in primitive endodermal cells (data not shown). When F9 cells were treated with T-RA and bt2cAMP to induce parietal endoderm differentiation, a selective depletion of TBP and TAFII135 also took place between day 3 and day 5 (Fig. lanes 1, 2 and 6), however, TBP, but not TAFII135, reappeared in parietal endodermal cell extracts around day 9 (lanes 4-5). Together the above results indicate that there is a specific regulation of TBP and TAFII135 during F9 cell differentiation.
Depletion of TBP and TAFII135 is not observed in F9 cells which are refractory to T-RA
To determine whether the depletion of TBP and TAFII
135 was intimately linked to the differentiation process, we examined the regulation of these proteins in mutant F9 cells with altered differentiation properties. F9 cells in which the retinoid X receptor α gene has been disrupted by homologous recombination (RXRα-/-
cells) are impaired in their response to T-RA [49
]. When these cells are treated with T-RA, the depletion of TBP and TAFII
135 observed in the cell extracts is delayed by almost 4 days and takes place only between days 7 and 9 rather than between days 3 and 5 in wild-type cells (Fig. ), while no such depletion is seen in extracts from vehicle treated cells (Fig. ). In contrast, no significant change in TBP and TAFII
135 is seen in extracts from T-RA treated RXRα-/-
cells, in which the RARγ gene has also been disrupted [50
] and which are known to be refractory to T-RA induced differentiation (Fig. ). Thus, the selective depletion of TBP and TAFII
135 requires that the F9 cells respond, at least partially, to T-RA, but does not require full differentiation to take place.
Figure 2 Depletion of TBP and TAFII135 is integral to the differentiation process. The positions of TBP and TAFII135 detected by immunoblotting are indicated. A. Extracts were prepared from mutant RXRα-/- cells differentiated with T-RA for the number of (more ...)
Depletion of TBP and TAFII135 is due to targeted proteolytic degradation
The depletion of TBP and TAFII135 in extracts from day 3-10 T-RA treated cells could result from transcriptional or post-transcriptional events. Semi-quantitative reverse-transcription PCR (RT-PCR) experiments using exon-specific oligonucleotide primers showed that there were no significant changes in the TAFII135 or TBP mRNA levels during differentiation (data not shown). This suggests that disappearance of TBP and TAFII135 in extracts from differentiated cells does not occur at the transcriptional level, but rather results from a post-transcriptional event.
The peptide aldehyde proteasome inhibitors MG132 and ALLN have previously been used to investigate the post-transcriptional regulation of proteins during physiological processes [52
]. To determine whether the depletion of TAFII
135 and TBP observed in the extracts from late T-RA treated F9 cells was due to their selective proteolysis, we treated T-RA differentiated cells with these inhibitors on days 3, 4, 5, 6, and 7. After an overnight exposure, extracts were prepared (day 4, 5, 6, 7 and 8 extracts in Figs. and ). In the absence of MG132 or ALLN, TBP and TAFII
135 disappeared in the differentiated cell extracts (middle panel Figs. and lanes 6-10). In contrast, in the presence of the inhibitors both proteins were readily detected even at late times where they are normally not observed (lanes 1-5). The levels were nevertheless lower than those detected in undifferentiated cell extracts. The level of TAFII
55, which remains constant during differentiation, is only mildly increased by the addition of MG132 or ALLN (Figs. and lower panel, lanes 1-5 and 6-10). The addition of MG132 or ALLN to non-differentiated cells also resulted in only a small increase in TBP and TAFII
135 levels (Figs. and , top panel compare lanes 1-5 and 6-10). The above results indicate that when proteolysis is inhibited the depletion of TBP and TAFII
135 is no longer observed showing that it is due to T-RA-induced proteolytic degradation.
Figure 3 Depletion of TBP and TAFII135 is abrogated in the presence of protease inhibitors MG132 and ALLN. A. Replica plates of cells were treated with T-RA or vehicle and after 3, 4, 5, 6, and 7 days MG132 was added overnight (12 hours) and extracts then prepared (more ...)
We also prepared extracts from differentiated cells where MG132 was added only at the time of extract preparation. Extracts made in this way are a measure of the intracellular level of these proteins since synthesis and degradation are both stopped at the same time. Comparison of the TBP and TAFII135 levels in extracts from day 7 differentiated cells prepared in the presence of MG132 showed that they were indeed significantly reduced compared to undifferentiated cells or day 3 differentiated cells (approximately 3 fold, Fig. , lanes 1, 4 and 5, similar results were obtained in the presence of ALLN data not shown). Nevertheless, these proteins do not disappear completely as they do in the absence of MG132 (lanes 2-3). Therefore, the complete disappearance of these proteins seen in the absence of protease inhibitors indicates that there is further degradation in the extracts which exaggerates the differences between differentiated and undifferentiated cells. Although, less TBP could be immunopurified from the MG132 blocked differentiated cell extracts using the anti-TBP antidody 2C1, when equal amounts of immunopurified TBP from differentiated and undifferentiated cells was loaded, equivalent amonts of TAFII135 were detected in the TFIIDs (data not shown). Thus, while the overall level of TFIID is diminished through the reduction in TBP and TAFII135, the relative stoichiometry of the remaining TFIID is not significantly altered. The apparently larger decrease in TAFII135 seen in Fig. results from the lower efficiency of the antibody (see also Fig. .)
Taken altogether, the above results show that the intracellular level of TBP and TAFII135 and consequently TFIID is significantly reduced in differentiated cells through T-RA-induced proteolytic degradation.
Coordinate degradation of TBP, TAFII135, and the RARgamma2 receptor during F9 cell differentiation
It has previously been shown that the RARγ2 is subject to degradation beginning 48 hours after T-RA-induced differentiation of F9 cells [42
]. To determine whether there is a coordinate degradation of RARγ2, TBP and TAFII
135, we examined extracts made at these early times. After 24 hours of T-RA treament the levels of RARγ2, TBP and TAFII
135 were unchanged (Fig. , lanes 1-2). However after 48 hours, there was a T-RA-dependent reduction in the amount of RARγ2 and TBP and a significant shift in the electrophoretic mobility of TAFII
135 (lanes 3-4). By 4 days the levels of all three proteins were strongly reduced (lanes 5-6). These results indicate that the onset of degradation of TBP and TAFII
135 is concomitant with that of the RARγ2 suggesting that these events are interdependent.
Constitutive ectopic expression of TAFII135 impairs F9 cell primitive endoderm differentiation
The above results show that the level of TAFII135 and TBP is strictly controlled during the differentiation of F9 cells by a post-transcriptional mechanism involving targeted proteolysis. To test whether artificially increasing the level of TAFII135 would affect the proper differentiation of F9 cells, we established cell lines which constitutively express flag-tagged versions of human TAFII135 [fTAFII135(372-1083) and fTAFII135(805-1083) see Materials and methods]. TAFII135(372-1083) contains all the known functional domains, but lacks an unconserved proline-alanine-rich region whereas, TAFII135(805-1083) comprises only the CR-II region.
For fTAFII135(372-1083) 12 cell lines were examined and we chose two, lines A and B, since they expressed the highest levels of the fTAFII135 protein. Immunoblotting with an anti-flag monoclonal antibody showed a significantly higher expression level in line A compared to line B (Fig. , lanes 1-2). This result is confirmed by immunoblotting with monoclonal antibody 20TA which detects both the endogenous and ectopically expressed TAFII135 proteins (Figs. and , lane 1). In line A, the level of the ectopically expressed protein is higher than that of the endogenous protein, while in line B both the exogenous and the endogenous proteins are expressed at similar levels. For fTAFII135(805-1083) which contains only the conserved C-terminal domain, 8 cell lines were examined of which 3 expressed the fTAFII135 protein (Fig. ). Both of the cell lines expressing fTAFII135(372-1083) showed an approximately 2-fold accelerated growth rate compared to wild type cells while no significant increase was seen in the fTAFII135(805-1083) expressing cells (Fig. , and data not shown, note that the apparent slowing of growth between day 8 and 10 for lines A and B is due to their reaching confluence).
Figure 4 Characterisation of F9 cell lines A and B expressing ectopic f TAFII135(372-1083). A. The presence of f TAFII135(372-1083) in extracts from each cell line was determined using the commercial anti-flag monoclonal antibody. B. Immunoblot analysis of extracts (more ...)
Figure 6 Isolation and analysis of cells expressing fTAFII135(805-1083). A. Extracts from wild-type cells and 5 different clones were analysed by immunoblotting using an anti-flag antibody. B Extracts were made from clone 2 cells differentiated with T-RA for the (more ...)
We first verified whether treatment of these cells with T-RA would lead to the depletion of the ectopically expressed TAFII135. In line A, both the ectopically expressed TAFII135 and the endogenous TAFII135 were depleted with the same kinetics (Fig. , lanes 1-5). TBP was also concomitantly depleted in these extracts, and all three proteins were stabilised when cells were treated with MG132 (lanes 6-8). Importantly however, the disappearance of all three proteins was clearly delayed by 48 hours compared to wild-type cells (compare Figs. and Fig. or Fig. ). In line B, there was also coordinate disappearance of TBP and the exogenous and endogenous TAFII135 (Fig. ), although no obvious delay such as that seen in line A is observed. In contrast, no depletion of fTAFII135(805-1083) was seen following T-RA treatment, while the endogenous TBP and TAFII135 were depleted normally between days 3 and 5 (Fig. , and data not shown).
As the targeted proteolysis of TBP and TAFII135 is linked to the differentiation process, the delayed depletion in line A suggests that T-RA induced differentiation may be perturbed. Their differentiation was therefore compared to that of the wild-type cells.
In wild type cells, the degradation of RARγ2 begins at day 2 and the protein is strongly depleted from extracts made at day 3 (Fig. , lane 3). At later times, low levels of only the unphosphorylated form of RARγ2 can be seen in the extracts (lanes 4-6). In contrast, in line A, elevated levels of RARγ2 are observed even at day 5 (Fig. , lanes 3-4). In line B, an intermediate situation is observed since high levels of RARγ2 are seen at day 3 which decrease by day 5 (lanes 3-4). In all cases, low levels of only the unphosphorylated RARγ2 are detected by days 7-9 (lanes 5-6). These results indicate that proteolytic degradation of RARγ2 is delayed in lines A and B suggesting that an early event in the differentiation process has been perturbed.
Comparison of the growth rate of line A with that of wild-type cells in the presence of T-RA, shows that line A is less sensitive to the anti-proliferative effect of T-RA than wild-type, however growth is eventually slowed compared to undifferentiated cells (Fig. ). Line B also grows faster than wild-type over the first 4-6 days, but subsequently its growth slows and is considerably retarded compared to undifferentiated cells (Fig. ). Together these observations show that line A is poorly sensitive to T-RA-induced growth arrest and retains an essentially undifferentiated morphology, while growth of line B slows after 4-6 days of T-RA treatment and morphological differentiation is retarded.
We determined whether fTAFII
135(372-1083) expression modified T-RA induced apoptosis. The proportion of early apoptotic cells in lines A, B, and wild-type was determined by flow cytometry in the presence of propidium iodide and annexin V-FITC [25
]. In wild-type cells, the proportion of early apoptotic cells increased from 13% in the absence of T-RA to 26% after 5 days of T-RA treatment (Fig. ). In line A, spontaneous apoptosis was lower than wild type (7% compared to 13%) and only a mild increase (9%) was seen by day 5 (Fig. ). In line B, spontaneous apoptosis was also lower than wild type (6% compared to 13%), while in the presence of T-RA the proportion rose to 16% (Fig. ). Thus, line A is resistant to T-RA induced apoptosis, while line B shows reduced sensitivity to T-RA induced apoptosis.
Detection of early apoptotic cells by flow cytometry. The percentage of early apoptotic cells in undifferentiated cultures and in day 5 T-RA-differentiated cultures is indicated for wild type cells and for lines A and B.
Differentiation of lines A and B was further assessed from changes in cellular morphology. 6-8 days after the addition of T-RA, wild-type F9 cells adopt a typical flattened primitive endodermal morphology (see upper panel of Fig. ). In contrast, line A cells retain an essentially undifferentiated morphology even after 8 days of T-RA treatment (centre panel Fig. ). The differentiation of line A is severely impaired, as the cells never adopt a differentiated morphology even after 10-12 days of RA-treatment (data not shown). Similarly, line B cells appear essentially undifferentiated after 6 days of T-RA treatment (lower panel, Fig. ). After 8 days, a fraction of the cells begin to adopt a primitive endodermal type morphology, although the cells are much less flattened than wild-type cells, and the majority of the cells retain an undifferentiated morphology (Fig. ). Only after 10 days of T-RA treatment did the majority of the cells adopt a differentiated morphology (data not shown).
Morphology of lines A and B in the presence of T-RA. A. Each panel shows representative phase-contrast photography at 125-fold magnification of wild-type cells or lines A and B after the indicated number of days treatment with T-RA.
F9 cell differentiation is characterised by the induction of different marker genes. We used reverse transcription coupled-PCR (RT-PCR) to compare the induction of some of these genes in lines A and B with that observed in wild-type cells. The RARβ2 gene is a marker which is rapidly induced in wild-type cells normally, after 12-24 hours [[49
]. In agreement with this, the RARβ2 mRNA is fully induced by day 3 in wild-type cells. Expression persists until day 5 and decreases only by day 7 (Fig. , lanes 2-5, note that the small increase in lane 5 in this experiment is not reproducibly observed). In contrast, in lines A and B, RARβ2 expression is weak at day 3, peaks only at day 5, and drops off by day 7 (lanes 7-10 and 12-15). Thus, induction of RARβ2 is severely retarded and transient in lines A and B. In the same RNA samples, constant expression levels of HPRT were observed in differentiated and undifferentiated cells. In contrast to the RARβ2 gene, induction of the collagen IVα1 and laminin B1 mRNAs was almost identical in the wild-type and mutant cells (Fig. and data not shown) showing that the delay in induction is specific to RARβ2.
Figure 8 RT-PCR analysis of gene expression during differentiation. A. Delayed induction of the RARβ2 gene. Aliquots of the PCR reactions made with RNA from the day shown above each lane were electrophoresed, blotted, and hybridised with an internal oligonucleotide (more ...)
After 6 days of T-RA treatment, bt2cAMP was added to induce parietal endoderm differentiation. Addition of bt2cAMP induced the rapid appearance of rounded cells with typical parietal endoderm morphology for both the wild type cells and lines A and B (Fig. ). Thus, while primitive endoderm differentiation of lines A and B is impaired, they readily differentiate into parietal endoderm.
Parietal differentiation of lines A and B. Each panel shows representative phase-contrast photography at 125-fold magnification of wild-type cells or lines A and B after 6 days of treatment with T-RA and two days with T-RA and bt2cAMP.
In lines A and B, we noticed after 6-8 days of T-RA treatment the appearance of a novel cell type characterised by elongated tightly packed cells (Fig. ). The morphology of these cells was completely different from those of undifferentiated or primitive endoderm cells. Although the majority of the cell population differentiates when bt2cAMP is added to these cultures, the elongated cells persisted even after several days of bt2cAMP treatment (Fig. ) showing that these cells are refractory to the differentiating effect of bt2cAMP. Cells with this atypical morphology were not observed when wild-type cells or fTAFII135(805-1083) expressing cells are differentiated either with T-RA or T-RA and bt2cAMP. Therefore, ectopic expression of fTAFII135(372-1083) has induced a novel differentiation pathway leading to the appearance of atypical elongated cells.
Figure 10 Abberant differentiation of lines A and B in the presence of T-RA. A. Phase contrast photograph of line B cells differentiated with T-RA showing the presence of elongated fibroblast-like cells. B. Phase contrast photograph of line A cells differentiated (more ...)
Taken altogether, the above results show that primitive endoderm differentiation of F9 cells is blocked in line A expressing the highest level of TAFII135(372-1083) and is significantly retarded in line B cells which express lower levels of TAFII135(372-1083). In contrast, all three fTAFII135(805-1083) expressing cell lines and several control lines picked at the same time all showed a normal T-RA response with respect to growth inhibition, differentiated cell morphology, and targeted depletion of TAFII135 and TBP (Fig. and data not shown).
TAFII135 and TBP are depleted in extracts from differentiated C2C12 cells
To determine whether the down regulation of TBP and TAFII
135 is specific to F9 cell differentiation we asked whether this phenomenon could be observed in an unrelated cell type. C2C12 are mouse skeletal muscle myoblasts which differentiate into multinucleated myotubes [57
]. This differentiation process does not require the addition of retinoic acid, but takes place spontaneously when cultures are grown to high density and undergo growth arrest.
In extracts from differentiating C2C12 cells, a progressive decrease in TAFII135 and TBP can be seen beginning on day 6 such that these proteins are strongly depleted from the extracts by day 11 (Fig. , lanes 1-5 compare with lane 6). In contrast, the levels of TAFII55 remain constant (Fig. ). The observed depletion of TBP and TAFII135 was not observed when the cells on day 10 were treated with MG132 for 12 hours prior to extract preparation on day 11 (Fig. , lanes 1-3). These results indicate that TBP and TAFII135 are also targetted for proteolytic degradation during C2C12 cell differentiation.
Figure 11 Depletion of TBP and TAFII135 in extracts from differentiated C2C12 cells. A. Immunoblot analysis of extracts from C2C12 cells grown at high density in the absence of serum for the number of days indicated above the panel. B. Immunoblot analysis of extracts (more ...)