1. Volume 14, Issue 2, Pages 175-182 (April 2004)
Gene-Specific Modulation of TAF10 Function by SET9-Mediated Methylation 
Antigone Kouskouti, Elisabeth Scheer, Adrien Staub, Làszlò Tora, Iannis Talianidis 
Molecular Cell 
Volume 14, Issue 2, Pages (April 2004)
DOI: /S (04)

2. Figure 1 SET9 Methylates TAF10 In Vitro at Lysine 189
(A) Baculovirus expression system-derived partial TFIID complex composed of TAF4, TAF5, TAF6, TAF8, TAF9, TAF10, and TAF12 (lane 1), or TAF6 and TAF9 (lane 2), were subjected to in vitro MTase reaction with 100 ng recombinant SET9.
(B) MTAse assays were performed with 100 ng SET9. As substrates, 10 μg of free histones, 1 μg of the indicated GST-TAF10 fusions, or His-tagged full-length TAF10 proteins were used. Numbers in the right panel indicate the positions of lysine residues mutated in the full-length His-TAF10 protein. The reaction products were analyzed by SDS-PAGE followed by autoradiography. The bottom panel (Coom. Blue) depicts part of the gel stained with Coomassie blue. The TAF10 amino acid sequence between positions 160 and 200 with highlights of the mutated lysine residues is depicted at the bottom.
(C) In vitro methylation reactions were performed using 2 μg of unmodified or lysine 189 mono- and dimethylated synthetic peptides corresponding to TAF10 (180–195 aa) and 100 ng SET9. The reaction products were analyzed by P81 filter binding assays. As control, a peptide synthesized from the C-terminal region of HNF-4 was used.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 TAF10 Interacts with SET9 and Is Methylated In Vivo
Methylated and unmethylated TAF10 incorporate into the TFIID complex in vivo.
(A) Nuclear extracts from HeLa cells were used for immunoprecipitations with preimmune serum or the monoclonal antibodies 1H8 (αTAF10-1), 2B11 (αTAF10-2), or 3G3 (αTBP) followed by Western blot analysis using the Pan-Methyl-Lysine antibody (Abcam).
(B) HeLa cell nuclei were incubated with 3H-SAM and after extract preparation were subjected to immunoprecipitations with the indicated antibodies and analyzed by SDS-PAGE. An autoradiogram of a 13 days exposure is shown.
(C) Nuclear extracts from Hela cells were immunoprecipitated with αSET9 and analyzed in Western blots with the indicated antibodies.
(D) Nuclear extracts from HEK293 cells transfected with the indicated expression vectors were immunoprecipitated with αHA. The precipitated proteins were analyzed in Western blots with the indicated antibodies.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3
The Methylation-Deficient Mutant TAF10 Can Rescue the Growth Defect of TAF10 Null F9 Cells but Compromises the Expression of ERF1 and ERA Genes
(A) F9:hTAF10(K189Q) cells were grown in the presence or absence of 1 μg/ml doxycyclin. Western blot analysis of whole-cell extracts was performed with TAF10 (2F4) antibody.
(B) Nuclei from F9:hTAF10wt cells (lanes 1 and 3) or F9:hTAF10(K189Q) cells (lanes 2 and 4) were incubated with 3H-SAM and after extract preparation were subjected to immunoprecipitation with αTAF10 (1H8) (lanes 1 and 2) or control antibody (lanes 3 and 4) and analyzed by Western blots with the antibodies shown at right. At the bottom, an autoradiogram of a 14 day exposure is shown.
(C) L−/L2TAF10 F9 cells harboring rtTA vectors were first electroporated with either phTAF10K189Q or yTAF10 linearized constructs. The cells grown in the presence of doxycycline were subcloned and then electroporated with pSG-Cre. The excision of exon 2 was monitored by PCR analysis of the genomic DNA. In lanes F9 and T, genomic DNA from wild-type F9 (F9) or F9:hTAF10 (T) cells was analyzed, respectively.
(D) RT-PCR analysis for the expression analysis of the indicated genes was performed with total RNAs prepared from F9:hTAF10 and F9:hTAF10(K189Q) cells before and after 5 days of treatment with retinoic acid (1 μM) and bt2cAMP (250 μM). Bars correspond to average signals from at least four experiments with independent RNA preparations.
(E) Chromatin immunoprecipitation (ChIP) analysis of SET9 and TAF10 occupancy and H3-K4-dimethylation of ERA, ERF1, cyclin E, and HPRT promoters in RA + cAMP-induced F9 cells. Bars represent real-time PCR values normalized to inputs and the values obtained with nonspecific antibody, which was set as one unit in each calculation.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
Involvement of TAF10 Methylation in SET9-Mediated Potentiation of Transcription
HEK293 cells were transfected with the indicated combinations of expression vectors and the appropriate luciferase reporter plasmids. Normalized luciferase activities and standard errors from four experiments are expressed as fold inductions above the activity obtained by Gal-TAF10wt (A), by the indicated activators without SET9 (B), and by Gal4-VP16 (C).
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5
SET9-Induced Methylation Increases the Affinity of the Interaction of TAF10 with RNA Polymerase II
(A) Extracts from HEK293 cells transfected with the indicated plasmids were immunoprecipitated with αHA antibody, and the presence of phosphorylated forms of RNA polymerase II in the precipitates was evaluated by Western blot analysis using H14 (Ser5-CTD) or H5 (Ser2-CTD) antibodies.
(B and C) Equal amounts (0.5 μg) of GST-TAF10wt or GST-TAF10K189Q were immobilized on glutathione agarose beads and incubated with 1 μg of recombinant SET9, in the absence (upper panels) or presence (lower panels) of 1 mM cold SAM for 1 hr at 30°C. The beads were then excessively washed with interaction buffer containing 500 mM NaCl to remove associated SET9, followed by incubation with HepG2 nuclear extracts. After washing, GST-TAF10-associated RNA polymerase II was monitored by Western blot analysis using the H14 antibody. Serial exposures of ECL images were quantitated by the NIH-image 1.63 software. Quantitative presentations of the data are depicted in the right panels.
Molecular Cell  , DOI: ( /S (04) )