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

Abstract

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 RARgamma2 receptor, and induction of target genes.

Results: We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAFII135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAFII135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAFII135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARgamma2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAFII135 delays proteolysis of the RARgamma2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAFII135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant.

Conclusions: These observations indicate that appropriately timed proteolysis of TBP and TAFII135 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.

Figure 1

A. Depletion of TBP and TAF_II135 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. 10 μg of each cell extract were then subjected to SDS-PAGE transferred to nitrocellulose membranes and the presence of TBP, and TAF_IIs was detected using the described antibodies. The locations of the detected proteins are indicated to the left. B. The extracts from cells differentiated for 0-9 days used in panel A were subjected to SDS-PAGE and stained with Coomasie Brilliant Blue. C. Western blot with anti-TBP and TAF_II135 antibodies performed on serial dilutions of an extract from undifferentiated F9 cells were compared with an undiluted extract from 7 day differentiated cells. Lanes 1-5 contain 20, 10, 4, 2, and 1 μg of undifferentiated cell extract and lane 6, 20 μg of differentiated cell extract. D. Immunoblotting shows that TAF_II135 and TBP are selectively depleted in F9 cell nuclear extracts. E. Extracts were prepared from F9 cells differentiated by the addition of T-RA and bt₂cAMP for the number of days indicated above each lane.

Figure 2

Depletion of TBP and TAF_II135 is integral to the differentiation process. The positions of TBP and TAF_II135 detected by immunoblotting are indicated. A. Extracts were prepared from mutant RXRα^-/- cells differentiated with T-RA for the number of days indicated above each lane. B. Control panel showing extracts from undifferentiated RXRα^-/- cells. C. Immunoblotting of extracts from T-RA treated RXRα^-/-RARγ^-/- cells.

Figure 3

Depletion of TBP and TAF_II135 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 designated day 4 to day 8 as indicated above each lane. TAF_II135, TBP and TAF_II55 were detected by immunoblotting. B. An experiment analogous to that shown in panel A was performed using the protease inhibitor ALLN. C. Measurement of the intracellular level of TAF_II135. In lanes 1-3 cell extracts were made in the absence of MG132. In lanes 4-5 cells were resuspended in extraction buffer containing 50 μM MG132. D. Coordinate degradation of TBP, TAF_II135 and the RARγ2. Cell extracts were made on the days indicated above each lane from T-RA treated or untreated cells and the presence of TBP, TAF_II135, and the RARγ2 was detected by immunobloting. The native RARγ and the phosphorylated species (pRARγ) are indicated along with a breakdown product indicated by ^*. A slower migrating species of TAF_II135 seen after 48 hours is indicated by -.

Figure 4

Characterisation of F9 cell lines A and B expressing ectopic f TAF_II135(372-1083). A. The presence of f TAF_II135(372-1083) in extracts from each cell line was determined using the commercial anti-flag monoclonal antibody. B. Immunoblot analysis of extracts from line A differentiated for the number of days indicated above each. Lanes 6-8 show extracts from cells treated for 12 hours with MG132. C. Immunoblot analysis of differentiated cell extracts from line B. D. Immunoblot analysis of extracts from wild-type cells and lines A and B with antibodies against RARγ2. The native and phosphorylated species and the breakdown product are indicated as in Fig. 4D. E. 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.

Figure 5

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.

Figure 6

Isolation and analysis of cells expressing fTAF_II135(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 number of days indicated above each lane. The fTAF_II135(805-1083) deletion mutant was detected in the cell extracts using the anti-flag antibody.

Figure 7

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.

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 probe for the RARβ2 gene. B. 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.

Figure 9

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.

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 with T-RA and bt₂cAMP. The rounded parietal endoderm cells can be seen alongside cells with an elongated morphology.

Figure 11

Depletion of TBP and TAF_II135 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 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.