Dataset Information

TAF8 MUTATION CAUSES INTELLECUAL DISABILITY, TFIID DISASSEMBLY WITH NO EFFECT ON RNA POLYMERASE II TRANSCRIPTION

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 nucleates RNA Polymerase II (Pol II) preinitiation complex formation on gene promoters and thus, is crucial for Pol II transcription. Germline knock out of several mouse TFIID subunits (Tbp, Taf7, Taf8, and Taf10) results in lethality at embryonic day 4.0, demonstrating the fundamental role of holo-TFIID in transcription. We identified a child harboring a splice-site mutation in TAF8, who has intellectual disability, poor growth, progressive spasticity and microcephaly. The c.781-G>A TAF8 mutation in this patient resulted in a frame shift, which affected the final 50 carboxy terminal amino acids of TAF8. We found that the mutant TAF8 protein is unstable and the patient c.781-G>A TAF8 primary fibroblasts did not form canonical TFIID complexes. Astonishingly however, genome-wide RNA pol II occupancy and pre-mRNA transcription on the tested genes was unaffected in the patient’s primary fibroblasts. This study indicates that perturbed TFIID function is less deleterious for transcription in human cells than originally anticipated.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

DISEASE(S): Intellectual Disability

SUBMITTER: Farrah El-Saafin

LAB HEAD: Laszlo Tora

PROVIDER: PXD008084 | Pride | 2018-04-16

REPOSITORIES: Pride

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Publications

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

El-Saafin Farrah F Curry Cynthia C Ye Tao T Garnier Jean-Marie JM Kolb-Cheynel Isabelle I Stierle Matthieu M Downer Natalie L NL Dixon Mathew P MP Negroni Luc L Berger Imre I Thomas Tim T Voss Anne K AK Dobyns William W Devys Didier D Tora Laszlo L

Human molecular genetics 20180601 12

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. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781- ...[more]

PMID: 29648665

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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.

Dataset Information

TAF8 MUTATION CAUSES INTELLECUAL DISABILITY, TFIID DISASSEMBLY WITH NO EFFECT ON RNA POLYMERASE II TRANSCRIPTION

Publications

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

Similar Datasets

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