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The TFIID subunit Taf13 is dispensable for RNA polymerase II transcription, but essential for early embryogenesis (ChIP-Seq)

ABSTRACT: RNA polymerase II transcription initiation requires assembly of a preinitiation complex (PIC). According to the stepwise model of PIC assembly, TFIID is the first GTF to be recruited. TFIID comprises TBP and 13 TBP-associated factors (TAFs). We focused on TAF13, the smallest subunit of TFIID. Taf13 has a histone-like domain that heterodimerizes with its partner Taf11. Recently published structures of TFIID place the Taf13-Taf11 heterodimer in lobe A where it forms a “bridge” between TBP and TFIID. According to this structure, the Taf11-Taf13 heterodimer has been suggested to be critical for TBP deposition at promoters. To better address this question, we generated mouse Embryonic Stem Cells (ESCs) cell lines and genetically modified mice where the gene coding for Taf13 was inactivated. Taf13-null mice died at E6.5-E7.5, whereas ESCs survived up to 15 days after inactivation of Taf13, but showed slower growth as compared to controls. To understand how Taf13 inactivation impacts TFIID and RNA polymerase II (Pol II) recruitment, we performed ChIP seq for the TBP and Taf1 subunits of TFIID along with PolII on ESC lines expressing or not Taf13. ChIP seq for histone marks H3K4Me3 and H3K27ac were also performed. Taf13 inactivation does not impair TBP recruitment at promoters, while that of Pol II was reduced both at the promoter and gene body. Surprisingly, recruitment of Taf1 was increased and while the H3K4Me3 mark was not affected, H3K27ac was notably increased in the absence of Taf13. Taken together, these data showed that loss of the Taf11-Taf13 heterodimer did not strongly impact TBP recruitment to promoters, but led to increased Taf1 and H3K27ac levels, but reduced paused and elongating Pol II.

ORGANISM(S): Mus musculus

PROVIDER: GSE254023 | GEO | 2025/06/18

REPOSITORIES: GEO

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Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells. Using homologous recombination, we generated ES cells and mice harbouring a null allele in the Tbp gene by insertion of a hygromycin resistance cassette in exon III and a floxed allele, in which exon III is surrounded by LoxP sites. From these mice, we generated an embryonic fibroblast Tbplox/- cell line in which TBP expression can be inactivated by expression of the Cre recombinase leading to cell death. We adopted a two-step strategy to generate Tbp-/- cell lines expressing human (h)TBP. Cells were first infected with pBABE retrovirus vectors expressing Wt hTBP or a series of mutants in the TBP core region, all of which carry a Flag-HA tag on their N-terminus. Cells expressing endogenous mTBP and exogenous hTBP were then infected with a second retrovirus expressing the 4 hydroxy-tamoxifen (OHT) inducible Cre-ERT2. Subsequently, multiple clonal populations from the OHT treated cells were isolated and genotyped by PCR to identify the Tbp-/-clones. At least two independent clones where expression of the endogenous mTBP was lost and replaced by the exogenous hTBP were isolated.

Project description:RNA polymerase II (Pol II) transcription initiation starts with the assembly of the preinitiation complex (PIC) on core promoters. The PIC is composed of six general transcription factors (GTFs). The recognition of the core promoter sequences by the TFIID GTF complex is the first step of the PIC assembly. In metazoans, holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Genetic depletion of different murine TAFs have shown that TAFs such as TAF7 or TAF10, can be either required, or dispensable for Pol II transcription, depending on different cellular contexts. In this report, we depleted TAF7 and/or TAF10 in the same cellular system; either in mesodermal progenitors during mouse development or in mESCs. In these two models, TAF7 depletion leads to a milder phenotype compared to TAF10 depletion. As TAF10 is also a subunit of the transcriptional co-activator Spt-Ada-Gcn5 acetyl transferase (SAGA), we first showed that the difference in phenotype between the Taf7 and Taf10 mutant is not due to the SAGA effect, at least for mESCs. Immunoprecipitations coupled with mass spectrometry analyses from mESCs lysates assembly of holo-TFIID complex is rapidly affected after induction of the depletion. In line with the model of holo-TFIID sequential assembly, TAF10 depletion leads to an early defect with formation of the core-TFIID, while TAF7 depletion results in the formation of a TAF7-less TFIID. Thus, the difference in phenotype severity correlates with the degree of TFIID disassembly. Surprisingly, no major global changes in Pol II transcription could be observed after either TAF7 or TAF10 depletion. Our data suggest that the inducible loss of fully assembled canonical TFIID does not correlate with the lack of global Pol II transcription activity changes suggesting that partially assembled TFIID complexes can participate in Pol II transcription initiation, with only limited effect on Pol II nascent transcription.

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The TFIID subunit Taf13 is dispensable for RNA polymerase II transcription, but essential for early embryogenesis (ChIP-Seq)

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