1. Identification of hTAFII80δ Links Apoptotic Signaling Pathways to Transcription Factor TFIID Function 
Brendan Bell, Elisabeth Scheer, Làszlò Tora 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1 Cloning of a Specialized Apoptotic Isoform of hTAFII80
(A) Schematic representation of the cDNAs of two putative splice variants of hTAFII80 and the open reading frames of the two isoforms they encode, hTAFII80α and hTAFII80δ. Narrow rectangles represent cDNA, and thick rectangles indicate protein sequences. An inverted Alu cassette inserted into hTAFII80δ is indicated by black shading, as are the α helices of the histone fold motif. Start codons (ATG) are indicated with arrows, and the epitopes used for antibody production are indicated by their names.
(B) A magnified view of the histone fold region shown in (A), showing the primary structure of the histone homology regions of hTAFII80α and hTAFII80δ, respectively.
(C) Apoptotic induction of hTAFII80δ mRNA expression. RT-PCR was used to detect the mRNAs encoding either hTAFII80δ or hTAFII80 in HeLa cells. RT-PCR products were transferred to nylon membranes and revealed by hybridization with radiolabeled oligonucleotide probes specific for all hTAFII80 isoforms except hTAFII80δ (lane 1) or probes for all known hTAFII80 isoforms including hTAFII80δ (lane 2).
(D) The ratio of hTAFII80δ mRNA with respect to that of hTAFII80α increases in HL-60 cells undergoing all-trans retinoic acid (RA)-induced apoptosis. RT-PCR products were obtained as in lane 2 of (A).
(E) Induction of hTAFII80δ mRNA by treatment of HL-60 cells with recombinant TRAIL. mRNA levels were measured by RT-PCR as in (C) and (D)
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
A Broad Spectrum of Apoptotic Stimuli Induce Caspase-Cleaved hTAFII80δ Levels
(A) HL-60 cells were treated with rhTRAIL (1 μM). Total cell extracts were prepared after no treatment (0 hr) or treatments for 2 hr or 6 hr and analyzed by SDS-PAGE followed by immunoblotting with antibodies raised against hTAFII80δ, hTAFII80α, and PARP. The cleavage products of hTAFII80δ and PARP are indicated by the subscript “cp.”
(B) HL-60 cells were treated with 1 μM rhTRAIL for 3 hr with (+) or without (−) pretreatment with 50 μM VAD-FMK for 30 min.
(C) As in (A), except treatment was with staurosporine (5 μM).
(D) As in (A), except treatment was with etoposide VP-16 (68 μM)
(E) As in (A), except treatment was with camptothecin (15 μM)
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
hTAFII80δ Expression Correlates with Apoptosis at the Single-Cell Level
(A) The nuclei of HeLa cells treated with etoposide VP-16 for 4 hr as revealed by DNA staining with Hoechst.
(B) Indirect immunofluorescent detection of hTAFII80δ using mAb 24TA (red).
(C) Immunofluorescent detection of a caspase cleavage product of cytokeratin 18 using FITC-labeled mAb M30 (green).
(D) Overlaid immunofluorescent signals from (B) and (C).
(E and F) hTAFII80 is ubiquitously expressed in HeLa cells. Nuclear Hoechst staining is shown in (E); indirect immunofluorescent detection of all known hTAFII80 isoforms is shown in (F)
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4
hTAFII80δ Is Present in a TFIID-like Complex Lacking hTAFII31 (TFIIDπ)
(A) Immunoprecipitation (IP) of endogenous hTAFII80δ and TBP-containing complexes from HeLa cells treated with etoposide VP-16 for 12 hr. Eluted complexes were concentrated 10-fold by trichloroacetic acid precipitation, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. hTAFII composition was determined by immunoblotting with the appropriate antibodies indicated at the left. As a negative control, mock IPs were performed by incubating protein-G beads alone with HeLa extract and processing them like IPs.
(B) Overexpressed hTAFII80δ incorporates into an hTAFII-containing complex that lacks hTAFII31. HeLa cells were transfected with a plasmid expressing hTAFII80δ. Total cell extracts were prepared and subjected to IPs with the indicated antibodies. Immune complexes were fractionated by SDS-PAGE and probed for TFIID components by immunoblotting. Input (lane 1) indicates the crude extract from transfected cells.
(C) Endogenous hTAFII80δ is not present in TFTC and is not found complexed with GCN5. IPs and immunoblots were performed as in (A); lane 3 contains purified TFTC (Wieczorek et al., 1998).
(D) hTAFII31 interacts in vitro with recombinant hTAFII80α but not with hTAFII80δ. Detection of recombinant TAFIIs from SF9 cells expressing Flag-hTAFII80α (lane 1), His-hTAFII80δ (lane 2), or hTAFII31 (lane 3) by immunoblotting with anti-TAFII80 mAb and anti-TAFII31Abs. Flag-hTAFII80α or His-hTAFII80δ-containing extracts were mixed with hTAFII31-containing extracts, followed by incubation with anti-Flag-agarose or Ni2+-agarose beads, respectively. After washing the beads, the amount of hTAFII31 bound to immobilized Flag-hTAFII80α (lane 4) or His-hTAFII80δ (lane 5) was analyzed by immunoblotting.
(E) A summary of protein-protein interactions observed between hTAFII80δ and hTAFII80α and other TAFIIs using coexpression in SF9 cells followed by coimmunoprecipitation
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
Expression of hTAFII80δ in HeLa Cells Causes Cell Death by Apoptosis
(A) Schematic representation of the hTAFII80δ and hTAFII80α and their derivatives used in transient transfection assays. For simplicity, domains have been given letter codes. The hTAFIIs reported to interact with various portions of hTAFII80 in vitro are indicated. The amino acids expressed by each of the deletion variants are given at the left and right of the schema. The unique N-terminal tail of hTAFII80δ is indicated by black shading, as are the α helices of the histone fold motif.
(B) Immunoblot analysis of transfected deletion variants. Arrowheads indicate overexpressed proteins.
(C) Percentage apoptosis induced by transfection of hTAFIIs as determined by flow cytometry. The standard deviation of three independent transfections is given by error bars. Positive values indicate increased apoptosis and negative values indicate inhibition of apoptosis.
(D) hTAFII31 coexpression rescues hTAFII80α-mediated but not hTAFII80δ-mediated apoptosis. Increasing amounts of hTAFII31 expression vector were cotransfected with a constant amount of hTAFII80δ or hTAFII80α expression plasmids. Apoptosis was measured as in (C) and expressed relative to the apoptosis induced by a given isoform in the absence of hTAFII31
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6
hTAFII80δ Selectively Alters Transcription by Targeting Cellular Promoters In Vivo
(A) Activation or repression of promoter-luciferase constructs by hTAFII80δ in transient cotransfection assays. Promoter-reporter constructs are shown on the left. Transfections were repeated three times with less than 15% variation in fold activation between experiments.
(B) Endogenous GADD45 and p21 protein levels are induced by hTAFII80δ overexpression. Levels of GADD45, p21, and MDM2 were monitored by immunoblotting total protein extracts from cells transfected with empty vector alone (lane 1) or hTAFII80δ (lane 2).
(C) Cooperative induction of endogenous p21 levels by hTAFII80δ and p53. HeLa cells were transfected as for the apoptosis assays. Cells were harvested 36–42 hr later, and total protein extracts were used for immunoblotting experiments. Transfected plasmids are indicated at the top of the panel.
(D) GADD45 levels correlate with hTAFII80δ induction during cell death induced by numerous stimuli. Endogenous GADD45 levels were measured by immunoblotting of cell extracts prepared from HL-60 cells after treatment for various times with apoptosis-inducing reagents as indicated.
(E) Occupancy of endogenous hTAFII80δ at cellular promoters in vivo. Chromatin immunoprecipitations were performed with the indicated mAbs on soluble chromatin prepared from unstimulated HeLa cells (−) or HeLa cells treated with 100 μM etoposide VP-16 for 6 hr (+). PCR amplification was used to determine the level of enrichment of promoter fragments in immunoprecipitated material
Molecular Cell 2001 8, DOI: ( /S (01) )