Abstract
The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.
Highlights
Transcription regulators in eukaryotes can be divided into three functional classes: gene-specific transcription regulators, cofactor complexes, and the general RNA polymerase transcription machinery
TAF8-TAF10 histone fold pair, together with one copy of the 5TAF core (TAF5-TAF6-TAF9-TAF4-TAF12) complex, (ii) TAF8 participates in connecting lobe B and C by interacting with the two HEAT repeats of TAF6 and certain regions of TAF1, and (iii) in lobe C the C-terminal half of TAF8 interacts with TAF2 (Fig. 1A) [12, 14, 18]
We focused on TAF8 interlinks where TAF8 cross-linked to another TATA box-binding protein (TBP)-associated factor (TAF) peptide and compared our dataset to cross-linking mass spectrometry (CXMS) data obtained from super core promoter (SCP)-bound hTFIID or SCP-bound hTFIID containing preinitiation complex (PIC) [12, 14] (Fig. 1B)
Summary
Transcription regulators in eukaryotes can be divided into three functional classes: gene-specific transcription regulators, cofactor complexes, and the general RNA polymerase transcription machinery. These crosslinking results in agreement with cryo-EM structures further indicate that TAF8 plays a triple role in TFIID: (i) participates in the assembly of the six HFD-containing 7TAF complex in lobe B via its N-terminus and HFD regions; (ii) connects lobes B and C by interacting with the HEAT repeat regions of two TAF6 subunits, the TAF1 region between amino acid (aa) 378 and 427, and TAF2 via its PRD; and (iii) interacts with TAF2 in lobe C via its C-terminal part.
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