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

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

Epigenetics & Chromatin ◽

10.1186/s13072-018-0180-6 ◽

2018 ◽

Vol 11 (1) ◽

Cited By ~ 7

Author(s):

Alejandro Saettone ◽

Jyoti Garg ◽

Jean-Philippe Lambert ◽

Syed Nabeel-Shah ◽

Marcelo Ponce ◽

...

Keyword(s):

Tetrahymena Thermophila ◽

Protein Complexes ◽

Related Protein ◽

Highly Expressed Genes

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Identification and Functional Characterization of Protest SWI/SNF and ISWI Complexes

10.32920/ryerson.14651697 ◽

2021 ◽

Author(s):

Alejandro Saettone Chipana

Keyword(s):

Chromatin Remodeling ◽

Tetrahymena Thermophila ◽

Affinity Purification ◽

Functional Characterization ◽

Peptide Array ◽

Interacting Proteins ◽

Silent Chromatin ◽

Lysine Residues ◽

Chromatin Remodeling Complexes

The thesis aims to identify and initiate functional characterization of the SWI/SNF and ISWI complexes in Tetrahymena thermophila. Through affinity purification of the conserved subunit Snf5 followed by mass spectrometry (AP-MS), I identified the first SWI/SNF complex in protists. One of the subunits I found is a small bromodomain containing protein named Ibd1. Through AP-MS of Ibd1 I found Ibd1 is versatile and interacts with several additional chromatin remodeling complexes. Bromodomains are known to have affinity for acetylated lysine residues within proteins such as histones. A peptide array experiment suggests that Ibd1 also has affinity for acetylated chromatin. Indirect immunofluorescence (IF) of Ibd1 hints at a role in transcription. My analysis of Tetrahymena Iswi1 shows expression during meiosis, vegetative growth and starvation. IF data shows its localization is consistent with Iswi1 function in mitosis/meiosis or maintenance of silent chromatin. AP-MS of ISW1 discovered several interacting proteins of unknown function.

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Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

10.32920/ryerson.14668284.v1 ◽

2021 ◽

Author(s):

Syed N Shah

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Chromatin Assembly ◽

Histone Chaperones ◽

Protein Protein Interactions ◽

Selective Forces ◽

Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

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How many remodelers does it take to make a brain? Diverse and cooperative roles of ATP-dependent chromatin-remodeling complexes in developmentThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o07-059 ◽

2007 ◽

Vol 85 (4) ◽

pp. 444-462 ◽

Cited By ~ 22

Author(s):

Elvin Brown ◽

Sreepurna Malakar ◽

Jocelyn E. Krebs

Keyword(s):

Chromatin Remodeling ◽

Dna Sequences ◽

Protein Complexes ◽

Peer Review Process ◽

Chromatin Remodelers ◽

Dna Accessibility ◽

Stage Of Development ◽

Chromatin Remodeling Complexes ◽

Cellular Machinery ◽

Selection Of

The development of a metazoan from a single-celled zygote to a complex multicellular organism requires elaborate and carefully regulated programs of gene expression. However, the tight packaging of genomic DNA into chromatin makes genes inaccessible to the cellular machinery and must be overcome by the processes of chromatin remodeling; in addition, chromatin remodeling can preferentially silence genes when their expression is not required. One class of chromatin remodelers, ATP-dependent chromatin-remodeling enzymes, can slide nucleosomes along the DNA to make specific DNA sequences accessible or inaccessible to regulators at a particular stage of development. While all ATPases in the SWI2/SNF2 superfamily share the fundamental ability to alter DNA accessibility in chromatin, they do not act alone, but rather, are subunits of a large assortment of protein complexes. Recent studies illuminate common themes by which the subunit compositions of chromatin-remodeling complexes specify the developmental roles that chromatin remodelers play in specific tissues and at specific stages of development, in response to specific signaling pathways and transcription factors. In this review, we will discuss the known roles in metazoan development of 3 major subfamilies of chromatin-remodeling complexes: the SNF2, ISWI, and CHD subfamilies.

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Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

10.32920/ryerson.14668284 ◽

2021 ◽

Author(s):

Syed N Shah

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Chromatin Assembly ◽

Histone Chaperones ◽

Protein Protein Interactions ◽

Selective Forces ◽

Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

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

10.32920/ryerson.14664858 ◽

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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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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