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

Abstract

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 associated with 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 decreased cellular viability and altered the expression of selected targets. 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.

Transcriptome analysis of the binucleate ciliate Tetrahymena thermophila with asynchronous nuclear cell cycles

“…In mammalian cells, MuvB complexes interact with E2F4/5, DP1/2 and an RB-like protein to generate transcriptional repressor complexes that inhibit expression of cell cycle regulated genes (Litovchick, Sadasivam et al DeCaprio (2007) Mol Cell; Sadasivam and DeCaprio, 2013, Nat Rev Cancer). In contrast, to the predicted Tetrahymena E2F repressors ( E2FL3 and E2FL4 ), none of the T. thermophila paralogues and Reb1 (Rb-like)-associated proteins identified by a proteomic-based approach (Nabeel-Shah, Garg et al 2021) are cell cycle regulated at the level of transcription (RebL1, Lin9, Lin54, Anqa1, Jinn1, Jinn2).…”

“…MLL1 is not periodically expressed. Similarly, genes responsible for macronuclear histone H3 demethylation ( THD1, SIN3/TTHERM_00450950, RXT2/TTHERM_00992830, SAP30, PHO23/TTHERM_000046389, SAP18/TTHERM_00469050 ) and histone H2 mono-ubiquitinylation ( RIN1/TTHERM_00263030; UB3 ligase) are not cell cycle regulated (Zhang, Gao et al 2014, Nabeel-Shah, Garg et al 2021).…”

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

“…Using this protocol, we have performed affinity purification followed by mass spectrometry analysis (AP-MS) to study FZZ-tagged nuclear proteins ( Saettone et al., 2019 ; Wahab et al., 2020 ) including histones ( Ashraf et al., 2019 ; Nabeel-Shah et al., 2020a ), histone chaperones, ( Garg et al., 2013 ; Nabeel-Shah et al., 2020b ; Saettone et al., 2018 ), and transcriptional regulatory Mediator-complex in Tetrahymena ( Garg et al., 2019 ). This protocol can also be used for identifying protein-protein interactions for cytoplasmic proteins.…”

KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

“…The AP-MS procedure was performed essentially as described (21,39). Briefly, two independent batches of ∼20x10 6 cells were grown representing two biological replicates.…”