Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila

Nabeel‐Shah, Syed; Garg, Jyoti; Tchara, Pata-Eting Kougnassoukou; Pearlman, Ronald E.; Lambert, Jean‐Philippe; Fillingham, Jeffrey

doi:10.1016/j.xpro.2021.100362

Cited by 3 publications

(4 citation statements)

References 23 publications

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“…Using our established pipeline ( 67 , 70 , 77 ), we performed affinity purifications (AP) coupled to tandem mass spectrometry (AP-MS) in biological replicates to identify the interaction partners of H4-FZZ. The MS data were scored using the SAINTexpress algorithm, which utilizes semiquantitative spectral counts for assigning a confidence value to individual protein-protein interactions (PPIs) ( 92 ).…”

Section: Resultsmentioning

confidence: 99%

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Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Nabeel‐Shah

Garg

Saettone

et al. 2021

Nucleic Acids Research

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Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

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Section: Resultsmentioning

confidence: 99%

“…For Tetrahymena samples, affinity purification was carried out essentially as described ( 67 , 70 ). Briefly, T. thermophila was grown in ∼500 mL of 1× SPP to mid-log phase to a final concentration of 3 × 10 5 cells/mL for vegetative samples.…”

Section: Methodsmentioning

confidence: 99%

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Nabeel‐Shah

Garg

Saettone

et al. 2021

Nucleic Acids Research

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“…We used our established affinity purification (AP) coupled to tandem mass spectrometry (AP-MS) pipeline to identify the H3 and H3.3 interactomes (Fig. 1B) [45,46]. The AP-MS data were curated using SAINTexpress which employs semiquantitative spectral counts for assigning a confidence value to individual protein-protein interactions [47].…”

Section: Identification Of Tetrahymena H3 and H33 Interaction Networkmentioning

confidence: 99%

“…Affinity purification was carried out essentially as described [45,46]. Tetrahymena cells were grown to midlog phase in ~ 500 ml of 1 × SPP to a final concentration of 3 × 10 5 cells/ml.…”

Section: Affinity Purification and Mass Spectrometry Sample Preparationmentioning

confidence: 99%

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

Nabeel‐Shah

Garg

Ashraf

et al. 2023

Epigenetics & Chromatin

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Background Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. Results We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Conclusion Our results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

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Tetrahymena

Cole¹

2022

Encyclopedia of Life Sciences

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The Tetrahymena genus is composed of mostly free‐living ciliated protozoa that have helped researchers gain insight into fundamental problems of cell biology. As all ciliates, Tetrahymena contain separate germline and somatic nuclei known as the micronucleus and macronucleus, respectively. The macronucleus is derived from a copy of the micronucleus through a process that involves extensive whole‐genome rearrangement and that is under intensive study. Tetrahymena thermophila can be readily manipulated using the tools of genetics, molecular biology, cell biology and biochemistry. Recently, next‐generation, whole‐genome sequencing has made possible the identification of previously unidentified genes responsible for a rich gallery of mutant phenotypes. Notable discoveries made using Tetrahymena include the structure of telomeres and telomerase, self‐splicing RNA, the first microtubular motor, and the link between histone acetylation and gene regulation. The approximately 104 Megabase macronuclear genome of T. thermophila has been sequenced and annotated; the micronuclear genome has been sequenced as well. Key Concepts Tetrahymena are large, complex eukaryotic cells well‐suited both for university research and for the undergraduate teaching‐laboratory. Tetrahymena, have been studied for nearly a century and have been the source of numerous ground‐breaking discoveries. Applying modern tools of genomics, proteomics, and next‐generation, whole‐genome sequencing has ushered in a new era of research to such topics as the assembly of basal bodies and cilia, pattern formation, novel roles for small ribonucleic acids ( RNAs ), membrane trafficking, secretion, phagocytosis, pinocytosis, cytoskeletal motility and chromosome dynamics including studies on the origins of deoxyribonucleic acid ( DNA ) replication, telomere synthesis, meiosis, recombination, apoptosis and the regulation of gene expression through epigenetic chromatin marking. Tetrahymena offer special insights into cell biological processes due to their many original and extraordinary capabilities including the use of a unique genetic code, novel twists on metabolism and protein trafficking, self‐splicing RNA, programmed genome rearrangement involved in genome surveillance, cortical patterning and cellular regeneration. Tetrahymena exhibit both conserved and evolutionarily divergent solutions to fundamental cell biological problems.

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Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila

Cited by 3 publications

References 23 publications

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

Tetrahymena

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