Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III

AbstractBackgroundThe relationship between transcription and the 3D genome organization is one of the most important questions in molecular biology, but the roles of transcription in 3D chromatin remain controversial. Multiple groups showed that transcription affects global Cohesin binding and genome 3D structures. At the same time, several studies have indicated that transcription inhibition does not affect global chromatin interactions.ResultsHere, we provide the most comprehensive evidence to date to demonstrate that transcription plays a marginal role in organizing the 3D genome in mammalian cells: 1) degraded Pol I, Pol II and Pol III proteins in mESCs, and showed their loss results in little or no changes of global 3D chromatin structures for the first time; 2) selected RNA polymerases high abundance binding sites-associated interactions and found they still persist after the degradation; 3) generated higher resolution chromatin interaction maps and revealed that transcription inhibition mildly alters small loop domains; 4) identified Pol II bound but CTCF and Cohesin unbound loops and disclosed that they are largely resistant to transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting RNA polymerases are capable of affecting the 3D genome indirectly. So, the direct and indirect effects of transcription inhibition explain the previous confusing effects on the 3D genome.ConclusionsWe conclude that Pol I, Pol II, and Pol III loss only mildly alter chromatin interactions in mammalian cells, suggesting the 3D chromatin structures are pre-established and relatively stable.

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RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011827 ◽

2020 ◽

Vol 295 (15) ◽

pp. 4782-4795 ◽

Cited By ~ 1

Author(s):

Philipp E. Merkl ◽

Michael Pilsl ◽

Tobias Fremter ◽

Katrin Schwank ◽

Christoph Engel ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase I ◽

Dna Templates ◽

Pol Ii ◽

Naked Dna ◽

Pol I ◽

Intrinsic Capacity ◽

Pol Iii ◽

Polymerase I

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

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A TATA-Binding Protein Mutant Defective for TFIID Complex Formation In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.3951 ◽

1999 ◽

Vol 19 (6) ◽

pp. 3951-3957 ◽

Cited By ~ 8

Author(s):

Ryan T. Ranallo ◽

Kevin Struhl ◽

Laurie A. Stargell

Keyword(s):

Binding Protein ◽

Tata Binding Protein ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Pol Ii ◽

Pol I ◽

Pol Iii ◽

Polymerase I ◽

Tfiid Complex

ABSTRACT Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L,K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.

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The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

Canadian Journal of Microbiology ◽

10.1139/m89-011 ◽

1989 ◽

Vol 35 (1) ◽

pp. 73-80 ◽

Cited By ~ 39

Author(s):

Wolfram Zillig ◽

Hans-Peter Klenk ◽

Peter Palm ◽

Gabriela Pühler ◽

Felix Gropp ◽

...

Keyword(s):

Nuclear Genome ◽

Distance Matrix ◽

Gene Organization ◽

Amino Acid Sequences ◽

Rna Polymerases ◽

Matrix Analysis ◽

Pol Ii ◽

Phylogenetic Relations ◽

Pol I ◽

Pol Iii

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A + C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial β′ and chloroplast β′ and β″ subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial β′ lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.Key words: transcription, evolution, taxonomy, subunits, gene organization.

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The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2644 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2644-2652 ◽

Cited By ~ 43

Author(s):

S D Brown ◽

J Huang ◽

L H Van der Ploeg

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Initiation Site ◽

Transcription Initiation Site ◽

Protein Coding ◽

Pol Ii ◽

Protein Coding Genes ◽

Pol I ◽

Pol Iii ◽

Repetitive Protein

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

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Analysis of the Caenorhabditis elegans rpc-1 gene

10.32469/10355/4129 ◽

2005 ◽

Author(s):

◽

Qun Zheng

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Rna Polymerase Iii ◽

Transgenic Animals ◽

Rna Polymerases ◽

Pol Ii ◽

C Elegans ◽

Pol I ◽

Genes Encoding ◽

Pol Iii

In eukaryotes, two large subunits form the core catalytic structure of RNA polymerase III (Pol III), which is conserved in other RNA polymerases, Pol I and Pol II. It has been found that Pol III activity is tightly associated to cell growth. TFIII B has been shown to be one of main mediators in this process. No regulation of the Pol III largest subunit gene has been found. In C. elegans, the rpc-1 gene encodes the largest subunit of Pol III. Here, I identified two critical structural components of RPC-1, Gly644 and Gly1055, whose mutations result in larval lethal arrestment. These two amino acid residues are universally conserved in RNA polymerases, indicating their overall involvement in gene transcription mechanism. Also, I found that maternally inherited, not embryonically expressed, rpc-1 gene products survive early development. Starvation was found to suppress rpc-1 gene expression and re-feeding treatment enhances rpc-1 gene expression rapidly. No similar regulation was detected in genes encoding largest subunits of Pol I and Pol II. This is the first time that rpc-1 gene regulation has been reported. Insulin signaling may not be involved in this regulation. Also, I found that rpc-1 promoter is not ubiquitously active in C. elegans. Using the rpc-1p::gfp transgene, the rpc-1 promoter activity is only detected in a subset of neurons in the head and the tail and the intestine. While starvation silences the rpc-1 promoter activity in most tissues and cells, ASK neurons still show GFP staining in the rpc-1p::gfp transgenic animals, indicating that rpc-1 transcription in ASK neurons is continuously active under starvation conditions. Further studies suggest that TGF-[beta] signaling is involved in mediating the rpc-1 promoter activity in ASK neurons.

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Chromatin interactions correlate with local transcriptional activity in Saccharomyces cerevisiae

10.1101/021725 ◽

2015 ◽

Author(s):

Zackary N. Scholl ◽

Jianling Zhong ◽

Alexander J. Hartemink

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Genome Organization ◽

Large Chromosome ◽

Transcription Rate ◽

Chromosome Conformation ◽

Pol Ii ◽

Chromatin Interactions ◽

Genome Wide ◽

Chromosome Segments

Genome organization is crucial for efficiently responding to DNA damage and regulating transcription. In this study, we relate the genome organization of Saccharomyces cerevisiae (budding yeast) to its transcription activity by analyzing published circularized chromosome conformation capture (4C) data in conjunction with eight separate datasets describing genome-wide transcription rate or RNA polymerase II (Pol II) occupancy. We find that large chromosome segments are more likely to interact in areas that have high transcription rate or Pol II occupancy. Additionally, we find that groups of genes with similar transcription rates or similar Pol II occupancy are more likely to have higher numbers of chromosomal interactions than groups of random genes. We hypothesize that transcription localization occurs around sets of genes with similar transcription rates, and more often around genes that are highly transcribed, in order to produce more efficient transcription. Our analysis cannot discern whether gene co-localization occurs because of similar transcription rates or whether similar transcription rates are a consequence of co-localization.

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Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

10.1101/339499 ◽

2018 ◽

Author(s):

Pratibha Bhalla ◽

Dipti Vernekar ◽

Ashutosh Shukla ◽

Benoit Gilquin ◽

Yohann Couté ◽

...

Keyword(s):

Rna Polymerase ◽

Associated Factors ◽

Rna Polymerase Iii ◽

Replication Stress ◽

Regulatory Proteins ◽

Pol Ii ◽

Adverse Conditions ◽

Pol I ◽

Paf1 Complex ◽

Pol Iii

AbstractMany regulatory proteins and complexes influence transcription by RNA polymerase (pol) II. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding genes. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to identify potential interactors through mass spectrometry-based proteomics. A large interactome constituting known chromatin modifiers, factors and regulators of transcription by pol I and pol II revealed the possibility of a large number of signaling cues for pol III transcription against adverse conditions. We found one of the pol II-associated factors, Paf1 complex (PAF1C) interacts with the three baits. Its occupancy on the pol III-transcribed genes is low and not correlated with pol III occupancy. Paf1 deletion leads to higher occupancy of pol III, γ-H2A and DNA pol2 but no change in nucleosome positions. Genotoxins exposure causes pol III but not Paf1 loss from the genes. PAF1C promotes the pol III pausing and restricts its accumulation on the genes, which reduces the replication stress caused by the pol III barrier and transcription-replication conflict on these highly transcribed genes.

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TATA-binding protein and associated factors in polymerase II and polymerase III transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7953 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7953-7960 ◽

Cited By ~ 15

Author(s):

R E Meyers ◽

P A Sharp

Keyword(s):

Associated Factors ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Pol Ii ◽

Transcription System ◽

Pol I ◽

Protein Functions ◽

Pol Iii ◽

Polymerase I

Transcription by RNA polymerase I (pol I), pol II, and pol III requires the TATA-binding protein (TBP). This protein functions in association with distinct TBP-associated factors (TAFs) which may specify the nature of the polymerase selected for initiation at a promoter site. In the pol III transcription system, the TBP-TAF complex is a component of the TFIIIB factor. This factor has been resolved into a TBP-TAF complex and another component, both of which are required for reconstitution of transcription by pol III. Neither the TBP-TAF complexes B-TFIID and D-TFIID, which were previously characterized as active for pol II transcription, nor TBP alone can complement pol III transcription reactions that are dependent upon the TBP-TAF subcomponent of TFIIIB. Surprisingly, the TBP-TAF subcomponent of TFIIIB is active in reconstitution of pol II transcription.

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SUMO and Transcriptional Regulation: The Lessons of Large-Scale Proteomic, Modifomic and Genomic Studies

Molecules ◽

10.3390/molecules26040828 ◽

2021 ◽

Vol 26 (4) ◽

pp. 828

Author(s):

Mathias Boulanger ◽

Mehuli Chakraborty ◽

Denis Tempé ◽

Marc Piechaczyk ◽

Guillaume Bossis

Keyword(s):

Large Scale ◽

Transcriptional Control ◽

Pol Ii ◽

Pol I ◽

New Concepts ◽

Gene Expression Control ◽

Genomic Studies ◽

Transcriptional Coregulators ◽

Pol Iii

One major role of the eukaryotic peptidic post-translational modifier SUMO in the cell is transcriptional control. This occurs via modification of virtually all classes of transcriptional actors, which include transcription factors, transcriptional coregulators, diverse chromatin components, as well as Pol I-, Pol II- and Pol III transcriptional machineries and their regulators. For many years, the role of SUMOylation has essentially been studied on individual proteins, or small groups of proteins, principally dealing with Pol II-mediated transcription. This provided only a fragmentary view of how SUMOylation controls transcription. The recent advent of large-scale proteomic, modifomic and genomic studies has however considerably refined our perception of the part played by SUMO in gene expression control. We review here these developments and the new concepts they are at the origin of, together with the limitations of our knowledge. How they illuminate the SUMO-dependent transcriptional mechanisms that have been characterized thus far and how they impact our view of SUMO-dependent chromatin organization are also considered.

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