Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Abstract

Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes. 135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant. 135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.

Background

Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes.

Results

135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant.

Conclusions

135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.

Background

]].

].

30 is not required for survival of retinoic acid differentiated F9 cells.

135 in more physiological situations.

].

135, TBP, and the RARγ2 in response to T-RA. The cells have an enhanced growth rate and their differentiation into primitive endoderm is impaired at an early stage, but they readily differentiate into parietal endoderm. Treatment of these cells with T-RA also induces the appearance of a population of cells with an atypical elongated morphology, distinct from that of the primitive endodermal cells, which have not been previously documented with wild type F9 cells, and which are resistant to differentiation with bt2cAMP.

135 are selectively depleted in extracts from T-RA differentiated F9 cells

]]. Differentiation is accompanied by growth inhibition, characteristic changes in cell morphology, targeted degradation of RARγ2, induction of marker genes, and increased apoptosis.

s, was used to monitor the levels of the respective proteins in differentiated cell extracts (see Materials and Methods).

). Therefore, there is a selective depletion of these two TFIID components in extracts from differentiating cells.

cAMP for the number of days indicated above each lane.

, 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).

).

135 during F9 cell differentiation.

135 is not observed in F9 cells which are refractory to T-RA

135 requires that the F9 cells respond, at least partially, to T-RA, but does not require full differentiation to take place.

cells.

135 is due to targeted proteolytic degradation

135 in extracts from differentiated cells does not occur at the transcriptional level, but rather results from a post-transcriptional event.

135 is no longer observed showing that it is due to T-RA-induced proteolytic degradation.

135 seen after 48 hours is indicated by -.

.)

135 and consequently TFIID is significantly reduced in differentiated cells through T-RA-induced proteolytic degradation.

135, and the RARgamma2 receptor during F9 cell differentiation

135 is concomitant with that of the RARγ2 suggesting that these events are interdependent.

135 impairs F9 cell primitive endoderm differentiation

135(805-1083) comprises only the CR-II region.

, 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).

. Cell growth was measured for 10 days in the presence or absence of T-RA. The cell number is indicated on the X axis and the number of days of growth is indicated on the Y axis. Analogous results were obtained in three independent experiments and the graph shows the results of a representative experiment.

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.

135(805-1083) deletion mutant was detected in the cell extracts using the anti-flag antibody.

, and data not shown).

135 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.

, 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.

). 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.

). Thus, line A is resistant to T-RA induced apoptosis, while line B shows reduced sensitivity to T-RA induced apoptosis.

). Only after 10 days of T-RA treatment did the majority of the cells adopt a differentiated morphology (data not shown).

. 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.

and data not shown) showing that the delay in induction is specific to RARβ2.

Aliquots of the PCR reactions performed with the same RNA samples as in panel A were analysed by electrophoresis on an agarose gel and staining with ethidium bromide.

). Thus, while primitive endoderm differentiation of lines A and B is impaired, they readily differentiate into parietal endoderm.

cAMP.

135(372-1083) has induced a novel differentiation pathway leading to the appearance of atypical elongated cells.

cAMP. The rounded parietal endoderm cells can be seen alongside cells with an elongated morphology.

and data not shown).

135 and TBP are depleted in extracts from differentiated C2C12 cells

]. 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.

135 are also targetted for proteolytic degradation during C2C12 cell differentiation.

. Immunoblot analysis of extracts from C2C12 cells in the presence or absence of MG132 added at day 10 and extracts prepared on day 11. Lanes 2 and 3 show duplicate extracts from two dufferent paltes treated with MG132.

135 and TBP in differentiated F9 cells

135 and TBP is a prerequisite for proper F9 celll differentiation.

135 is observed further confirming that this is indeed a post-transcriptional event.

135 and consequently TFIID are reduced in differentiated cells through selective proteolysis.

135 in differentiating F9 cells. In this respect we note that we have not been able to recover F9 cell lines significantly overexpressing TBP suggesting that TBP overexpression is toxic.

135 in response to T-RA indicates that the cellular T-RA response is not only regulated through degradation of the activator, but also through degradation of components of the basal transcription machinery.

135 is also observed when C2C12 cells differentiate. Hence, targeted degradation can be induced by stimuli other than T-RA. Our report is the first showing that basal transcription factors can be regulated in this way in response to different physiological stimuli.

135 are modified directly to allow targeting, or whether they are targeted indirectly through the formation of transcriptionally active complexes with the RARγ2 which, as it is itself degraded, acts to recruit the degradation machinery.

135. This pathway may therefore be used to more generally regulate the activity of all three RNA polymerases during differentiation.

135 plays a key role in F9 cell growth and differentiation

135(805-1083) differentiate normally.

cells where none of these events take place.

135 is only targeted for degradation if it contributes functionally to the T-RA response.

135 expression modulates the activity of transcription factors in addition to the RARs leading to the appearance of this novel cell type.

Conclusions

135 at elevated levels promotes cell growth, impairs the normal T-RA response, and induces a novel differentiation pathway.

F9 and C2C12 cell culture and differentiation

cells and after 4-10 days cells were counted with a particle counter (Coulter Z2). C2C12 cells were maintained undifferentiated by growth at low density. C2C12 cells were differentiated by plating at high density and growth in 0.1% serum for the number of days indicated on the figures. Morphological differentiation was clearly seen by light microscopy.

Extract preparation and immunoblotting

]. Anti-flag monoclonal antibody was puchased from Sigma. Blots were revealed using peroxidase conjugated goat anti-mouse IgG antibody and chemiluminescence with an ECL kit (Amersham). Protease inhibitors MG132 (Cbz-leu-leu-leucinal) and ALLN (acetyl-leu-leu-norleucinal) were purchased from CalBiochem and ICN Biochemicals respectively. Each was resuspended in DMSO and added to cells or extracts at a final concentration of 50 μM.

RT-PCR and flow cytometry

cells per sample were analysed with the CELLQuest software.

Acknowledgements

We thank D. Metzger and F.Cammas for providing the RXR/RAR mutant cell lines and for advice on F9 cell culture and differentiation, C. Egly for help with RARγ2 and discussion, C. Waltzinger, for help with flow cytometry, M. Abrink and Y-G. Gangloff for critical reading of the manscript, S. Vicaire and D. Stephane for DNA sequencing, the monoclonal antibody facility, the staff of cell culture and oligonucleotide facilities, B. Boulay, J.M. Lafontaine, and C. Werlé for illustrations, L.P was supported by a Marie Curie EU fellowship and E.K. by fellowships from the Fondation Chateaubriand and INSERM. This work was supported by grants from the CNRS, the INSERM, the Hôpital Universitaire de Strasbourg, the Ministère de la Recherche et de la Technologie, the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and the Human Frontier Science Programme.