Zinc knuckle of TAF1 is a DNA binding module critical for TFIID promoter occupancy

Abstract

The general transcription factor IID (TFIID) is the first component of the preinitiation complex (PIC) to bind the core promoter of RNA polymerase II transcribed genes. Despite its critical role in protein-encoded gene expression, how TFIID engages promoter DNA remains elusive. We have previously revealed a winged-helix DNA-binding domain in the N-terminal region of the largest TFIID subunit, TAF1. Here, we report the identification of a second DNA-binding module in the C-terminal half of human TAF1, which is encoded by a previously uncharacterized conserved zinc knuckle domain. We show that the TAF1 zinc knuckle aids in the recruit of TFIID to endogenous promoters vital for cellular proliferation. Mutation of the TAF1 zinc knuckle with defects in DNA binding compromises promoter occupancy of TFIID, which leads to a decrease in transcription and cell viability. Together, our studies provide a foundation to understand how TAF1 plays a central role in TFIID promoter binding and regulation of transcription initiation.

Figure 1

TAF1 contains an evolutionarily conserved zinc knuckle. (A) Linear schematic of full length human TAF1 with percent conservation in eukaryotes shown. (B) Annotated TAF1 zinc knuckle conservation alignment spanning vertebrates, insects, nematodes, plants, and fungi. Zinc knuckle cysteines and histidine are boxed in blue. Conserved positive residues indicated by the green dots. (C) Sequence alignment of zinc knuckle containing proteins from eukaryotes and viruses. (D) Alignment of TAF1 ZnK model (blue) from I-TASSER prediction analysis with known structure of ZnK of HIV-1 nucleocapsid protein (green) and lin28 (purple); (E) Electrostatic surface map of HIV-1 nucleocapsid protein nucleic acid binding surface (right) and corresponding region of TAF1 ZnK (left).

Figure 2

TAF1 zinc knuckle is required for cell viability. (A) Phase contrast images of ts13 cells transfected with pCS2 + (vector), wild type TAF1 (WT), winged-helix mutant (WH3A), zinc knuckle mutant containing two cysteine to alanine mutations (ZnM), and double mutant (3AZnM) at 39.5 °C for 72 hrs. (B) Quantitation of viable DAPI stained cells (n = 3). Error bars represent standard deviation. Two-tailed analysis compared to vector with a 95% confidence, ***p < 0.0001 (Unpaired t-test). (C) Western blot of ts13 lysates expressing HA-tagged TAF1 proteins. Proteins were immunoprecipitated with TAF1 specific double bromodomain antibody and immunoblotted for exogenously expressed TAF1 using anti-HA antibody. The full-length blot is presented in Supplemental Figure S4.

Figure 3

TAF1 zinc knuckle and winged helix are imperative for effective cyclin promoter activity. (A) Incorporation of TAF1 proteins into TFIID. TFIID complexes were isolated by immunoprecipitation and incorporated HA-TAF1 variants detected by anti-HA immunoblotting. Additional TFIID subunits immunoprecipitated were detected by immunoblotting. The full-length blots are presented in Supplemental Figure S5. Quantitation of normalized HA-TAF1 and TAF protein levels are provided in Supplemental Table 2. (B) Chromatin immunoprecipitation of HA-TAF1 variants expressed in HEK293 cells followed by qPCR for cyclin D1 and cyclin A2 promoters (n = 4). (C) Luciferase assay of cyclin D1 and cyclin A2 promoter driven reporter constructs co-transfected with TAF1 variants. Luciferase activity was normalized for total protein and expressed relative to reporter activity without exogenous TAF1 (Empty), given a value of 1.0 (n = 3). All error bars represent standard deviation. Two-tailed analysis compared to WT with a 95% confidence, **p < 0.01, ***p < 0.0001 (Unpaired t-test).

Figure 4

TAF1 zinc knuckle binds DNA. (A,B,C) EMSA of TAF1 ZnA (aa 1234–1375) and three radiolabeled DNA fragments: IMD of super core promoter (position −6 to +38), cyclin D1 promoter (position −22 to +29, CD1P), and Random DNA sequence. (D,E,F) Bio-layer interferometry binding curves using biotinylated double-stranded DNA fragments described in above and the following ZnA protein concentrations: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, 12 nM. Raw data was plotted with GraphPAD Prism. K_d was calculated from plotting steady-state binding levels against protein concentration.

Figure 5

Core Module and Key Residues in TAF1 Zinc Knuckle DNA Binding Domain. (A) TAF1 (aa 1234–1375) was incubated without (upper) and with (lower) IMD promoter DNA followed by digestion with increasing concentrations of protease. Digestion products were resolved by SDS-PAGE and detected by coomassie blue staining. Arrowheads indicate fragments stabilized by DNA. Quantification of protein fragments are provided in Supplemental Figure S3. (B) EVfold map analysis of ZnA. Numbers represent amino acid residues of full-length TAF1. (C) Diagram of DNA stabilized regions of TAF1 with blue box indicating CCHC ZnK. DNA binding curves for (D) ZnC wild type, (E) ZnC cysteine mutant (C1285A and C1288A), (F) ZnC charge mutant (R1295A and K1298A), (G) ZnD wild type, (H) ZnD cysteine mutant, and (I) ZnD charge mutant. Raw data was plotted with GraphPAD Prism. K_d was calculated by plotting steady-state binding levels against protein concentration and determining the concentration needed for half maximal binding.

Figure 6

Model of TAF1’s Role in Promoter Recognition. (A) ZnK and WH Bind Simultaneously. (B) Sequential Binding of ZnK and WH.