Genetic and Proteomic Analysis of Rna Polymerase Ii-Interacting Complexes Mediator and Integrator in the Ciliated Protozoan Tetrahymena Thermophila

10.32920/ryerson.14664858.v1 ◽

2021 ◽

Author(s):

Matthew D. R. Cadorin

Keyword(s):

Rna Polymerase Ii ◽

Tetrahymena Thermophila ◽

Slow Growth ◽

Protein Complexes ◽

Affinity Purification ◽

Model Systems ◽

Ciliated Protozoan ◽

Conventional Model ◽

Colony Pcr ◽

Independent Functions

In most eukaryotes, the largest subunit of RNAPII, Rpb1, contains a conserved carboxyterminal domain (CTD) containing a canonical structure of heptapeptide repeats. Two protein complexes of interest, Mediator and Integrator, are known to interact with this CTD in all eukaryotic models they have been described in to date. Recently, orthologs of Mediator and Integrator subunits have been identified within the ciliated protozoan Tetrahymena thermophila; one of the few eukaryotic lineages to lack a canonically organized CTD. To begin to characterize putative Mediator and Integrator complexes within T. thermophila, I engineered appropriate macronuclear tagging and knockout cassettes. Although the Tetrahymena MED31 ortholog was unable to rescue the slow growth phenotype of a yeast MED31 knockout, or co-purify with yeast Med8-TAP, I identified subunit Med3 as a member of the Med31 interactome in T. thermophila through tandem affinity purification coupled with mass spectrometry. I also targeted the Tetrahymena INTS6 locus for knockout as determined by colony PCR. If Mediator and Integrator exist in Tetrahymena despite its divergent CTD of Rpb1, perhaps these complexes have CTD-independent functions beyond what can be effectively studied using conventional model systems.

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bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila

10.32920/14640522.v1 ◽

2021 ◽

Author(s):

Alejandro Saettone ◽

Jyoti Garg ◽

Jean-Philippe Lambert ◽

Syed Nabeel-Shah ◽

Marcelo Ponce ◽

...

Keyword(s):

Tetrahymena Thermophila ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Methyl Transferase ◽

Chromatin Remodelers ◽

Highly Expressed Genes ◽

Chromatin Remodeling Complexes ◽

Additional Protein ◽

Active Transcription

Background The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. Results Using affinity purification coupled to mass spectrometry (AP–MS) we identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt, two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits localize exclusively to the transcriptionally active macronucleus during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP–MS results identified a BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during growth but not development. AP–MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP–MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth. Conclusions Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin-remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.

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Establishment of Proximity-dependent Biotinylation Approaches in Different Plant Model Systems

10.1101/701425 ◽

2019 ◽

Cited By ~ 2

Author(s):

Deepanksha Arora ◽

Nikolaj B. Abel ◽

Chen Liu ◽

Petra Van Damme ◽

Klaas Yperman ◽

...

Keyword(s):

Structural Information ◽

Protein Complexes ◽

Affinity Purification ◽

Lotus Japonicus ◽

Model Systems ◽

Test Case ◽

Plant Model ◽

One Step ◽

Subsequent Capture ◽

Biotin Labelling

AbstractProximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and allows subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of PDL in plants. Here we present the results of a systematic study applying a variety of PDL approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.

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The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila

10.32920/14640522.v2 ◽

2021 ◽

Author(s):

Alejandro Saettone ◽

Jyoti Garg ◽

Jean-Philippe Lambert ◽

Syed Nabeel-Shah ◽

Marcelo Ponce ◽

...

Keyword(s):

Tetrahymena Thermophila ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Methyl Transferase ◽

Chromatin Remodelers ◽

Highly Expressed Genes ◽

Chromatin Remodeling Complexes ◽

Additional Protein ◽

Active Transcription

Background The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. Results Using affinity purification coupled to mass spectrometry (AP–MS) we identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt, two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits localize exclusively to the transcriptionally active macronucleus during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP–MS results identified a BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during growth but not development. AP–MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP–MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth. Conclusions Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin-remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.

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The bromodomain-containing protein Ibd1 links multiple chromatin-related protein complexes to highly expressed genes in Tetrahymena thermophila

10.32920/14640522 ◽

2021 ◽

Author(s):

Alejandro Saettone ◽

Jyoti Garg ◽

Jean-Philippe Lambert ◽

Syed Nabeel-Shah ◽

Marcelo Ponce ◽

...

Keyword(s):

Tetrahymena Thermophila ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Methyl Transferase ◽

Chromatin Remodelers ◽

Highly Expressed Genes ◽

Chromatin Remodeling Complexes ◽

Additional Protein ◽

Active Transcription

Background The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. Results Using affinity purification coupled to mass spectrometry (AP–MS) we identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt, two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits localize exclusively to the transcriptionally active macronucleus during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP–MS results identified a BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during growth but not development. AP–MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP–MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth. Conclusions Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin-remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.

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Affinity Tags for Protein Purification

Current Protein and Peptide Science ◽

10.2174/1389203721666200606220109 ◽

2020 ◽

Vol 21 (8) ◽

pp. 821-830

Author(s):

Vibhor Mishra

Keyword(s):

Protein Purification ◽

Protein Complexes ◽

Affinity Purification ◽

Protein Domain ◽

Affinity Tag ◽

Small Peptides ◽

Affinity Tags ◽

C Terminus ◽

Solubility Enhancers ◽

As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

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Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.737 ◽

1996 ◽

Vol 142 (3) ◽

pp. 737-747 ◽

Cited By ~ 7

Author(s):

Jacques Archambault ◽

David B Jansma ◽

James D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Steady State ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Reporter Gene ◽

Growth Medium ◽

Temperature Sensitivity ◽

Slow Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

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Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes

Journal of Proteomics ◽

10.1016/j.jprot.2014.09.011 ◽

2015 ◽

Vol 118 ◽

pp. 81-94 ◽

Cited By ~ 148

Author(s):

Jean-Philippe Lambert ◽

Monika Tucholska ◽

Christopher Go ◽

James D.R. Knight ◽

Anne-Claude Gingras

Keyword(s):

Protein Complexes ◽

Affinity Purification ◽

Interactome Mapping

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X-ray and UV Radiation Damage of dsDNA/Protein Complexes

Molecules ◽

10.3390/molecules26113132 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3132

Author(s):

Paweł Wityk ◽

Dorota Kostrzewa-Nowak ◽

Beata Krawczyk ◽

Michał Michalik ◽

Robert Nowak

Keyword(s):

Dna Damage ◽

Simple Model ◽

Uv Radiation ◽

Protein Complexes ◽

Chemical Processes ◽

Model Systems ◽

Degradation Pathways ◽

X Ray ◽

Physico Chemical ◽

Sensitization Process

Radiation and photodynamic therapies are used for cancer treatment by targeting DNA. However, efficiency is limited due to physico-chemical processes and the insensitivity of native nucleobases to damage. Thus, incorporation of radio- and photosensitizers into these therapies should increase both efficacy and the yield of DNA damage. To date, studies of sensitization processes have been performed on simple model systems, e.g., buffered solutions of dsDNA or sensitizers alone. To fully understand the sensitization processes and to be able to develop new efficient sensitizers in the future, well established model systems are necessary. In the cell environment, DNA tightly interacts with proteins and incorporating this interaction is necessary to fully understand the DNA sensitization process. In this work, we used dsDNA/protein complexes labeled with photo- and radiosensitizers and investigated degradation pathways using LC-MS and HPLC after X-ray or UV radiation.

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Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6303 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6303-6310 ◽

Cited By ~ 37

Author(s):

L Yu ◽

M A Gorovsky

Keyword(s):

Protein Sequence ◽

Tetrahymena Thermophila ◽

Histone H3 ◽

Constitutive Expression ◽

Histone Variants ◽

Wild Type ◽

Ciliated Protozoan ◽

Knockout Mutant ◽

Double Knockout ◽

Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

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