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Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID

ABSTRACT: Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunits basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID is built using co-translational assembly. We discovered that all early steps of TFIID assembly, involving protein heterodimerization, happen during protein synthesis. Strikingly, we identify TAF1 – the largest protein in the complex – as a flexible scaffold subunit that co-translationally recruits preassembled TFIID submodules found populating the cytoplasm of cells. Consequently, TAF1 depletion leads to a cytoplasmic accumulation of TFIID building blocks. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly on nascent TAF1 polypeptide, that works as a ‘driver’ subunit. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

INSTRUMENT(S): Orbitrap Exploris 480, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Luc Negroni

LAB HEAD: Laszlo Tora

PROVIDER: PXD036358 | Pride | 2023-07-04

REPOSITORIES: Pride

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Genome-wide transcriptional dependencies on TAF1 functional domains

Project description:TFIID plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TAF subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast (Saccharomyces cerevisiae) TAF1 reportedly possesses at least four distinct activities including a histone acetyltransferase, and TBP, TAF, and promoter binding. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature sensitive taf1ts2 yeast strain. After galactose-induction and temperature inactivation of the temperature-sensitive allele, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise ~90% of the yeast genome, displayed a strong dependency upon all regions of TAF1 that were tested. This might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes. Keywords: genetic modification

2006-01-10 | GSE3006 | GEO

Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription.

Project description:TFIID is a central player in activated transcription initiation. Recent evidence suggests that the role and composition of TFIID is more diverse than previously understood. To investigate the effects of changing the composition of TFIID in a simple system we depleted TAF1 from Drosophila cells and determined the consequences on metal induced transcription at an inducible gene, Metallothionein B (MtnB). We observe a marked increase in the levels of both the mature message and pre-mRNA in TAF1 depleted cells. Under conditions of continued metal exposure, we show that TAF1 depletion increases the magnitude of the initial transcription burst, but has no effect on the timing of that burst. We also show that TAF1 depletion causes delay in the shut-off of transcription upon removal of the stimulus. Thus TAFs are involved in both establishing an upper limit of transcription during induction and efficiently turning the gene off once the inducer is removed. Using genomewide nascent-seq we identify hundreds of genes that are controlled in a similar manner indicating that the findings at this inducible gene are likely generalizable to a large set of promoters. There is a long-standing appreciation for the importance of the spatial and temporal control of transcription. Here we uncover an important third dimension of control, the magnitude of the response. Our results show that the magnitude of the transcriptional response to the same signaling event, even at the same promoter, can vary greatly depending on the composition of the TFIID complex in the cell. Nascent RNA was sequenced from replicate samples of Drosophila S2 cells treated with double-stranded RNA directed against E. coli LacI (Control) or against Drosophlia TAF1 (experimental). Reads per kilo-base per million (RPKM) was determined for each gene and the control and experimental samples were compared to determine the genes that were affected by the depletion of TAF1.

2013-05-01 | E-GEOD-45873 | biostudies-arrayexpress

SRRM4/nSR100 directs neuronal-specific microexon splicing of TAF1 mRNA involved in X-linked Dystonia-Parkinsonism

Project description:X-linked dystonia-parkinsonism is a neurodegenerative disease, which is caused by a SVA retrotransposon insertion within TAF1, gene encoding an integral component of the basal transcription factor TFIID. The SVA insertion has been shown to induce defects both in biosynthesis and in alternative splicing of TAF1 mRNA in various cell types. This includes the reduction of a neuron-specific isoform of TAF1 mRNA generated by inclusion of the evolutionary conserved microexon 34’ (TAF1-34’). In this study, we investigated the tissue distribution of TAF1-34’ mRNA and protein and the neuron-specific mechanism sustaining its alternative splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34’ have different distributions in the brain. To our knowledge, this is the first in situ detection of a microexon. We find that the differential expression of these two isoforms distinguishes proliferating from post-mitotic neurons in vitro and in vivo. Knockdown and ectopic expression experiments in cell lines demonstrated that the neuron-specific splicing factor nSR100/SRRM4 is directing the inclusion of microexon 34’ into TAF1 mRNA. These results show that SRRM4 regulates temporal and spatial distribution of alternative TAF1 mRNAs to generate a neuron-specific isoform of basal transcription factor TFIID.

2020-05-26 | PXD009905 | Pride

Genome-wide relationships between TAF1 and histone acetyltransferases in S. cerevisiae - chIP-chip study

Project description:Evidence suggests that the TAF1 subunit of TFIID is a histone acetyltransferase (HAT) that is functionally redundant with the Gcn5 HAT of the SAGA and ADA complexes. Here we test a number of predictions of this hypothesis by examining the in vivo histone acetylation targets of TAF1 and Gcn5, and re-examining the basis for the reported genome-wide functional redundancy between TAF1 and Gcn5. Our findings do not support a number of basic tenets of the hypothesis, thus bringing into question the physiological presence of any TAF1 HAT function in yeast. We have also conducted genome-wide expression profiles of numerous other HATs (Elp3, Hat1, Hpa2, Sas3) in an effort identify potential functional redundancy between TAF1 and other HATs, and find none. Further investigation of TAF1 and the Esa1 HAT re-affirm a link between histone H4 acetylation by Esa1, and TFIID binding via interactions with acetylated histone H4-binding protein Bdf1. Keywords: ChIP-chip, genetic modification

2006-03-20 | GSE3346 | GEO

Genome-wide relationships between TAF1 and histone acetyltransferases in S. cerevisiae

2006-03-20 | GSE3345 | GEO

Native holdup (nHU) to measure binding affinities from cell extracts

Project description:Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

2023-03-11 | PXD036024 | Pride

Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID

Project description:Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We reexamined this longstanding problem by rapid depletion of TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Tafs 1,2,7,11, and 13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing and TATA-less gene classes. The magnitude of TFIID-dependence varies with growth conditions, although this variation is similar at all gene classes. Many studies have suggested differences in gene regulatory mechanisms between TATA and TATA-less genes and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID contributes significantly to expression of nearly all yeast mRNAs and that differences in regulation between these two gene classes is due to other properties.

2017-08-02 | GSE97081 | GEO

HELZ2: a new, interferon-regulated, human 3’-5’ exoribonuclease of the RNB family is expressed from a non-canonical initiation codon

Project description:Proteins containing a RNB domain, originally identified in E. coli RNase II, are widely present throughout the tree of life. Many RNB proteins are endowed with 3’-5’ exoribonucleolytic activity but some have lost catalytic function during evolution. Database searches identified a new RNB domain containing protein in human: HELZ2. Analysis of genomic and expression data with evolutionary information suggested that the human HELZ2 protein is produced from an unforeseen non-canonical initiation codon in Hominidae. This unusual property was confirmed experimentally, extending the human protein by 247 residues. Human HELZ2 was further shown to be an active ribonuclease despite the substitution of a key residue in its catalytic center. HELZ2 harbors also two RNA helicase domains and several zinc-fingers and its expression is induced by interferon treatment. We demonstrate that HELZ2 is able to degrade structured RNAs through the coordinated ATP-dependent displacement of duplex RNA mediated by its RNA helicase domains and its 3’-5’ ribonucleolytic action. The expression characteristics and biochemical properties of HELZ2 support a role for this factor in response to viruses and/or mobile elements.

2023-08-18 | PXD040351 | Pride

Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly

Project description:TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit composition exist and several TFIID subunits are shared with other complexes, conveying intricate cellular decision making to control subunit allocation and functional assembly of this essential transcription factor. However, the underlying molecular mechanisms remain poorly understood. Here, we used quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5/TAF6/TAF9 submodule identified novel interactions crucial for TFIID integrity, and for allocating TAF9 to TFIID or the SAGA co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6/TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are crafted in the cell, involving checkpoint decisions facilitated by a chaperone machine.

2019-01-07 | PXD011293 | Pride

Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly

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