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Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery [ChIP-seq]

ABSTRACT: A great deal of work has revealed in structural detail the components of the machinery responsible for mRNA gene transcription initiation. These include the general transcription factors (GTFs) which assemble at promoters along with RNA Polymerase II (Pol II) to form a preinitiation complex (PIC) aided by the activities of cofactors and site-specific transcription factors (TFs). However, less well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining on a mechanistic level how rates of in vivo RNA synthesis are established and how cofactors and TFs impact them. We used competition ChIP to obtain genome-scale estimates of the residence times for five GTFs: TBP, TFIIA, TFIIB, TFIIE and TFIIF in budding yeast. While many GTF-chromatin interactions were short-lived (< 1 min), there were numerous interactions with residence times in the several minutes range. Sets of genes with shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism and therefore offer a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription. The relationships between gene function and GTF dynamics suggest that shared sets of TFs tune PIC assembly kinetics to ensure appropriate levels of expression. Scripts and processed fileas are available from: https://github.com/AubleLab/PIC_competition_ChIP_scripts

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE235000 | GEO | 2023/07/27

REPOSITORIES: GEO

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Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery [RNA-seq]

Project description:A great deal of work has revealed in structural detail the components of the machinery responsible for mRNA gene transcription initiation. These include the general transcription factors (GTFs) which assemble at promoters along with RNA Polymerase II (Pol II) to form a preinitiation complex (PIC) aided by the activities of cofactors and site-specific transcription factors (TFs). However, less well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining on a mechanistic level how rates of in vivo RNA synthesis are established and how cofactors and TFs impact them. We used competition ChIP to obtain genome-scale estimates of the residence times for five GTFs: TBP, TFIIA, TFIIB, TFIIE and TFIIF in budding yeast. While many GTF-chromatin interactions were short-lived (< 1 min), there were numerous interactions with residence times in the several minutes range. Sets of genes with shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism and therefore offer a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription. The relationships between gene function and GTF dynamics suggest that shared sets of TFs tune PIC assembly kinetics to ensure appropriate levels of expression. Scripts and processed fileas are available from: https://github.com/AubleLab/PIC_competition_ChIP_scripts

2023-07-27 | GSE235001 | GEO

Requirements for preinitiation complex formation in vivo

Project description:Transcription by RNA polymerase (Pol) II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the core promoter. In vitro, the TATA-binding protein (TBP), TFIIB, TFIIF, and Pol II are essential for PIC formation and transcriptional initiation, whereas TFIIA, TFIIE, and TFIIH are not required under certain conditions. In addition, the PIC is stable in the absence of nucleotide triphosphates, and sequential addition of GTFs results in a series of partial PICs. Here, we analyze the roles of all GTFs in yeast cells by measuring PIC formation and Pol II transcription upon depletion of individual GTFs. All GTFs are essential for TBP binding and Pol II transcription in vivo, suggesting that partial PICs composed of GTF subsets do not exist at appreciable levels. In contrast, TBP-associated factors (TAFs) contribute to Pol II transcriptional activity at most (and perhaps all) genes, but TAF-independent transcription occurs at a substantial level, particularly at promoters lacking canonical TATA elements. Lastly, unlike the case in vitro, PICs are not observed in cells deprived of uracil, and presumably UTP, suggesting a mechanism that removes transcriptionally inactive PICs from promoters.

2019-02-27 | GSE122734 | GEO

Effects of sumoylation-impairing mutation of TFIIF subunit Tfg1 on genome-wide RNA polymerase II (RNAPII) levels in budding yeast

Project description:Many transcription-related proteins, including components of the RNA polymerase II (RNAPII) general transcription factors (GTFs), have been shown through proteomics studies to be modifiable by SUMO conjugation. However, we determined that the large subunit of TFIIF, Tfg1, is likely the major target of sumoylation among GTFs in budding yeast. The primary site of sumoylation on Tfg1 is Lys residues 60 or 61, which lies in a region of the protein that is proximal to the Rpb2 subunit of RNAPII when the GTF-RNAPII preinitiation complex (PIC) assembles at promoters. As TFIIF and RNAPII are highly functionally and physically linked, we examined whether blocking Tfg1 sumoylation through Lys-to-Arg mutations could affect the recruitment or association of RNAPII with chromatin. RNAPII ChIP-seq was performed in strains expressing wild-type (Tfg1-HA) or sumoylation-deficient (Tfg1-K60,61R-HA) forms of Tfg1. Indeed, nearly half of all protein-coding genes showed significantly reduced RNAPII levels in the mutant strain. These data suggest that sumoylation of TFIIF plays an important role in regulating the dynamics of RNAPII association with genes.

2021-09-21 | GSE167426 | GEO

ChIP-Chip of General Transcription factors in Halobacterium NRC-1

Project description:Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes. In bacteria sigma factors regulate much of this process while in eukaryotes four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, using a systems approach, we provide experimental evidence (including protein-coIP, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions. We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs. This report represents a significant contribution towards closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to utilize various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little is previously known. Keywords: ChIP-Chip

2007-02-23 | GSE7045 | GEO

Nucleolar TFIIE plays a role in ribosomal biogenesis and performance

Project description:Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE – due to impaired ribosomal biogenesis and translational accuracy – lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.

2021-11-16 | PXD023451 | Pride

RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex [ChIP-seq]

Project description:RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.

2022-05-04 | GSE193679 | GEO

RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex [ChIP-Rx]

2022-05-04 | GSE193678 | GEO

RPAP2 regulates a transcription initiation checkpoint by prohibiting assembly of preinitiation complex [TT-seq]

2022-05-04 | GSE192782 | GEO

An RNA Polymerase III General Transcription Factor Engages in Cell Type-Specific Chromatin Looping

Project description:Transcription factors (TFs) bind DNA in a sequence-specific manner and have been proven to serve as anchors to the 3D genome organization. These TFs are generally cell type-specific factor and/or developmental master regulators. General TFs (GTFs) which serve RNA polymerase recruitment to specific promoter sequences, however, can be part of very large protein complexes but their roles in genome architecture have not been carefully explored. Here, we used a bio-informatic approach of ChIP-seq and Hi-C data analysis to depict the role of one GTF of the RNA polymerase III system (TFIIIC) in several human cell lines, both normal and transformed. We find that TFIIIC occupancy within the genome mainly occurs at specific regions which largely correspond to Alu elements but also at characteristic classes of repetitive elements (REs) such as MIR, FLAMC and ALR/alpha, depending on the cell’s developmental origin. We then show that these TFIIIC-enriched regions are involved in cell type-specific DNA looping within the genome and do not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge about the role of TFIIIC as a bona fide genome organizer whose action can lead to cell type-dependent 3D genome looping via binding to REs.

2022-02-22 | GSE195499 | GEO

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters [ChIP-Exo]

Project description:Genome-wide, little is understood about how proteins organize at inducible promoters before and after in-duction, and to what extent inducible and constitutive architectures depend on cofactors. We report that se-quence-specific transcription factors and their tethered cofactors (e.g., SAGA, Mediator, TUP, NuA4, SWI/SNF, RPD3-L) are already bound to promoters prior to induction (“poised”), rather than recruited upon induction, whereas induction recruits the pre-initiation complex (PIC). Through depletion and/or deletion ex-periments we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 does acetylate +1 nucleosomes there. At poised promoters, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. At induced promoters, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID to stably associate, rather than creating a TFIID-independent PIC, as is generally thought. These findings suggest that inducible systems, where present, evolved on top of constitutive systems.

2022-10-27 | GSE212654 | GEO

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