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. 2021 Nov;297(5):101288.
doi: 10.1016/j.jbc.2021.101288. Epub 2021 Oct 9.

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival

Elisabeth Scheer  1 , Jie Luo  2 , Andrea Bernardini  1 , Frank Ruffenach  1 , Jean-Marie Garnier  1 , Isabelle Kolb-Cheynel  1 , Kapil Gupta  3 , Imre Berger  3 , Jeff Ranish  2 , László Tora  4
Affiliations

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival

Elisabeth Scheer et al. J Biol Chem. 2021 Nov.

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.

Keywords: CRISPR/Cas9; TAF8; TATA-binding protein (TBP); TBP-associated factors (TAFs); TFIID; embryonic stem cells (ESCs); function; knock out; structure; viability.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
TAF8 interactions in human TFIID.A, the human TFIID cryo-electron microscopy model is shown. The model used in the panel is the super core promoter (SCP)-bound TFIID-TFIIA in post TBP-loading state (12), PDB: 7EGJ. TAF8 is highlighted in rose. TAF8 positions used in our study and visible in the TFIID structure are labeled with rose circles with their corresponding amino acid positions highlighted in white. TBP, TFIIA, and TAFs in the TFIID complex are indicated with their corresponding colors. Lobe B is composed of TAF5, 4–12, 6–9 and TAF8–10, and Lobe C of TAF1, TAF2, and TAF7. The HEAT repeats of the two TAF6s connecting the three lobes of TFIID are highlighted. Lobe A is hidden for clarity (lays behind lobe C from this view). B, TAF8 interlinks (cross-links between TAF8 and other TFIID subunits) identified by CXMS analyses of our endogenous TFIID complex preparation. All cross-links were identified with high confidence and the number of times a cross-linked peptide was identified is indicated in brackets (Tables S1 and S2). Upper part, CXMS done in this study, lower part, CXMS done by Patel et al. (2018) or Chen et al. (2021). Lysines (K) in TAF8 and the residues to which they cross-link on other TAFs are indicated. Green line indicates the TAF8 path visible in the cryo-EM structure (12).
Figure 2
Figure 2
Characterization of TAF8 interactions within the TAF2-TAF8-TAF10 subcomplex.A, schematic presentation of human TAF8, its domain structure, and the different TAF8 deletions. N: N-terminal domain; HFD: histone fold domain; ID: intermediary domain; PRD: proline-rich domain; and T2R1, T2R2, and T2R3: TAF2-interacting regions 1, 2, and 3. B–D, TAF2 and TAF10 were coexpressed with either WT TAF8 or with TAF8 deletions as indicated above each lane using the Baculovirus overexpression system. Input extracts (B) and anti-TAF10 IPs (C) were tested by western blot analyses (WB) with the indicted antibodies. D, anti-TAF10 IPs were also tested by silver-staining (silver) of the gels. TAF8 and its deletions are indicated with an arrowhead. On panels (B–D) the molecular weight markers (M) are indicated in kDa. D, IgG H: heavy chain of the antibody. On panels (B–D) (lane 1) indicates Sf9 cell extract without any coinfection. IgG H, heavy chain of the antibody; NS, nonspecific band.
Figure 3
Figure 3
Characterization of TAF8 interactions within the 7TAF complexes.A and B, TAF5-TAF6-TAF9-TAF4-TAF12 and TAF10 were coexpressed with either WT TAF8 or with TAF8 deletions as indicated above each lane using the Baculovirus overexpression system. Anti-TAF10 IPs (A) were tested by western blot analyses (WB) with the indicted antibodies, or by silver-staining (silver) of the gels (B). On panel (A) the molecular weight markers (M) are indicated in kDa. B, the arrow heads indicate TAF8 or the different TAF8 deletions. IgG H: heavy chain of the antibody. The western blot assay tests corresponding to the TAF expressions in the Input extracts for (A and B) are shown in Fig. S3. C and D, the substitution of human TAF8 117–140 amino acid sequences with the S. cerevisiae Taf8 sequences does not rescue 7TAF complex formation. TAF5-TAF4-TAF12-TAF6-TAF9 and TAF10 were coexpressed with either WT TAF8, or with the TAF8 substitution in which the human 117 to 140 amino acid (aa) sequences were replaced with the yeast 156 to 179 aa sequences ([TAF8(Y156–179)] as indicated above each lane) using the Baculovirus overexpression system. Input extracts (C) or anti-TAF10 IPs (D) were tested by western blot analyses (WB) with the indicted antibodies. Molecular weight markers (M) are indicated in kDa. -: indicates Sf9 cell extract without any coinfection. Dotted lines indicate where the gels were cut. NS, nonspecific band.
Figure 4
Figure 4
Characterization of TAF8 interactions within the 8TAF complexes.A and B, TAF2-TAF5-TAF6-TAF9-TAF4-TAF12 and TAF10 were coexpressed with either WT TAF8 or with TAF8 deletions, as indicated above each lane, using the Baculovirus overexpression system. Anti-TAF10 IP elutions (A) were tested by western blot analyses (WB) with the indicted antibodies, or by silver-staining (silver) of the gel (B) as described in Figure 3, A and B. The molecular weight markers (M) are indicated in kDa. B, the arrow heads indicate TAF8 or the different TAF8 deletions. Dotted line indicates where the gel was cut. Input extracts for the anti-TAF10 IPs are shown in Figure S3. IgG H, heavy chain of the antibody; NS, nonspecific band.
Figure 5
Figure 5
Generation of ESCs exogenously expressing Flag-TAF8, Flag-TAF8Δ117–140, and Flag-TAF8Δ141–184.A–C, stable doxycycline (+Dox)-inducible E14 mESC populations were generated in which WT R:Flag-mTAF8 (A), R:Flag-mTAF8Δ117–140 (B), or R:Flag-mTAF8Δ141–184 (C) were exogenously expressed. Stable ESCs were isolated, whole cell extracts (WCE) prepared and tested by western blot assay for the expression of the corresponding Flag-tagged TAF8 proteins using an anti-Flag antibody (as indicated). D–F, ESC clones originating from one single ESC, expressing the individual Flag-mTAF8 (D), Flag-mTAF8Δ117–140 (E), or Flag-mTAF8Δ141–184 (F) proteins were amplified in presence of Dox (+), WCEs prepared, and the expression of the corresponding TAF8 proteins (as indicated) was tested by western blot assay using an anti-TAF8 antibody. Endogenous (endo) TAF8 is also labeled. Individual ESC clones are labeled A to M (D), A to L (E) and 13 to 23 (F), and the ESC clones selected for further analyses are labeled in red. Ponceau staining was used as loading control in all panels. Molecular weight markers (M) are indicated in kDa. Dotted lines indicate where the gels were cut in (A and C).
Figure 6
Figure 6
The exogenously expressed Flag-TAF8Δ117–140 and Flag-TAF8Δ141–184 deletions are impaired in TFIID assembly in mESCs.A, exogenously and endogenously expressed TAF8 proteins were compared in the selected Flag-mTAF8(A+), Flag-mTAF8Δ117–140(A+), Flag-mTAF8Δ141–184(21+) clones (see Fig. 5, D–F) and wild-type (WT) ESC WCEs by western blot analysis using an anti-TAF8 antibody. The different Flag tagged TAF8 proteins are labeled with small arrows and the position of endogenous (endo) TAF8 is shown by an arrow on the right. Ponceau staining was used as loading control in all panels. Molecular weight markers (M) are indicated in kDa. B and C, mass spectrometry analysis of either anti-Flag (B) or anti-TAF7 IPs (C) carried out using WCEs (B) or nuclear extracts (C) prepared from ESCs expressing Flag-TAF8, Flag-mTAF8Δ117–140, Flag-mTAF8Δ141–184, or WT ESCs. Three technical replicates were carried out. Normalized spectral abundance factor (NSAF) values were calculated and normalized to the bait of the IPs (to Flag-tagged TAF8 or its deletions, in B; and to TAF7 in C). The normalized NSAF results are represented as heat maps with the indicated scales. TFIID subunit identification details are in Table S5.
Figure 7
Figure 7
The expression of Flag-TAF8Δ117–140 or Flag-TAF8Δ141–184 does not rescue the knockout of the endogenous Taf8 gene in mESCs.A, schematic representation of the mouse Taf8 locus, with exons and introns and with the direction of transcription shown. The genomic positions of the mouse Taf8 locus on chromosome 17 (chr17) are indicated. The positions of the two gRNAs, surrounding exon 2 used to knockout the endogenous Taf8 locus, are indicated. B, table showing the numbers of individual viable ESC clones (R:Flag-TAF8, R:Flag-TAF8Δ117–140 or R:Flag-TAF8Δ141–184) screened following the CRISPR/Cas9 KO of the endogenous Taf8 gene. Percentages of the obtained heterozygous (+/−) and homozygous ESC clones (−/−) are indicated. C, schematic illustration depicting how TAFs are thought to interact in holo-TFIID (without DNA). The three lobes of TFIID are labeled A to C. TAFs are labeled by their corresponding numbers and the same color code as used Figure 1A. HFDs or HFD-containing TAFs are depicted as circles. TAF8 regions connecting lobe B and C are shown as a magenta line or cylinder. Some regions of TAF1 are labeled with a dotted line. Domains and connecting lines are not to scale.
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References

    1. Fuda N.J., Ardehali M.B., Lis J.T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature. 2009;461:186–192. - PMC - PubMed
    1. Roeder R.G. The complexities of eukaryotic transcription initiation: Regulation of preinitiation complex assembly. Trends Biochem.Sci. 1991;16:402–408. - PubMed
    1. Thomas M.C., Chiang C.M. The general transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 2006;41:105–178. - PubMed
    1. Muller F., Tora L. Chromatin and DNA sequences in defining promoters for transcription initiation. Biochim. Biophys. Acta. 2014;1839:118–128. - PubMed
    1. Tora L. A unified nomenclature for TATA box binding protein (TBP)-associated factors (TAFs) involved in RNA polymerase II transcription. Genes Dev. 2002;16:673–675. - PubMed

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