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1.  The TFIID Components Human TAFII140 and Drosophila BIP2 (TAFII155) Are Novel Metazoan Homologues of Yeast TAFII47 Containing a Histone Fold and a PHD Finger 
Molecular and Cellular Biology  2001;21(15):5109-5121.
The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAFIIs). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAFIIs and overall molecular organization. In recent years, it has been assumed that all the metazoan TAFIIs have been identified, yet no metazoan homologues of yeast TAFII47 (yTAFII47) and yTAFII65 are known. Both of these yTAFIIs contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAFII25. We have cloned a novel mouse protein, TAFII140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAFII140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAFII155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAFII47 as their HFDs selectively heterodimerize with dTAFII24 and human TAFII30, metazoan homologues of yTAFII25. We further show that yTAFII65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAFII. These conserved domains are critical for yTAFII65 function in vivo. Our results therefore identify metazoan homologues of yTAFII47 and yTAFII65.
doi:10.1128/MCB.21.15.5109-5121.2001
PMCID: PMC87236  PMID: 11438666
2.  Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells 
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
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 RARγ2 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 RARγ2 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.
doi:10.1186/1471-2199-2-4
PMCID: PMC31370  PMID: 11285139
3.  The Human TFIID Components TAFII135 and TAFII20 and the Yeast SAGA Components ADA1 and TAFII68 Heterodimerize to Form Histone-Like Pairs 
Molecular and Cellular Biology  2000;20(1):340-351.
It has been previously proposed that the transcription complexes TFIID and SAGA comprise a histone octamer-like substructure formed from a heterotetramer of H4-like human hTAFII80 (or its Drosophila melanogaster dTAFII60 and yeast [Saccharomyces cerevisiae] yTAFII60 homologues) and H3-like hTAFII31 (dTAFII40 and yTAFII17) along with two homodimers of H2B-like hTAFII20 (dTAFII30α and yTAFII61/68). However, it has not been formally shown that hTAFII20 heterodimerizes via its histone fold. By two-hybrid analysis with yeast and biochemical characterization of complexes formed by coexpression in Escherichia coli, we showed that hTAFII20 does not homodimerize but heterodimerizes with hTAFII135. Heterodimerization requires the α2 and α3 helices of the hTAFII20 histone fold and is abolished by mutations in the hydrophobic face of the hTAFII20 α2 helix. Interaction with hTAFII20 requires a domain of hTAFII135 which shows sequence homology to H2A. This domain also shows homology to the yeast SAGA component ADA1, and we show that yADA1 heterodimerizes with the histone fold region of yTAFII61/68, the yeast hTAFII20 homologue. These results are indicative of a histone fold type of interaction between hTAFII20-hTAFII135 and yTAFII68-yADA1, which therefore constitute novel histone-like pairs in the TFIID and SAGA complexes.
PMCID: PMC85089  PMID: 10594036
4.  Synergistic Transcriptional Activation by TATA-Binding Protein and hTAFII28 Requires Specific Amino Acids of the hTAFII28 Histone Fold 
Molecular and Cellular Biology  1999;19(7):5050-5060.
Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAFII28) with the altered-specificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D3 from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the α2-helix of the histone fold in the conserved C-terminal region of hTAFII28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAFII18. This α-helix of hTAFII28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1′ α-helix of TBP either reduces or increases interactions with hTAFII28. The mutations which reduce interactions with hTAFII28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAFII28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAFII28.
PMCID: PMC84343  PMID: 10373554

Results 1-4 (4)