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

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.

Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila.

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.

Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in Tetrahymena thermophila Reveals an Ancient Network of Chaperones.

Epigenetic information, which can be passed on independently of the DNA sequence, is stored in part in the form of histone posttranslational modifications and specific histone variants. Although complexes necessary for deposition have been identified for canonical and variant histones, information regarding the chromatin assembly pathways outside of the Opisthokonts remains limited. Tetrahymena thermophila, a ciliated protozoan, is particularly suitable to study and unravel the chromatin regulatory layers due to its unique physical separation of chromatin states in the form of two distinct nuclei present within the same cell. Using a functional proteomics pipeline, we carried out affinity purification followed by mass spectrometry of endogenously tagged T. thermophila histones H2A, H2B and variant Hv1.We identified a set of interacting proteins shared among the three analyzed histones that includes the FACT-complex, as well as H2A- or Hv1-specific chaperones. We find that putative subunits of T. thermophila versions of SWR- and INO80-complexes, as well as transcription-related histone chaperone Spt6Tt specifically copurify with Hv1. We also identified importin ß6 and the T. thermophila ortholog of nucleoplasmin 1 (cNpl1Tt) as H2A-H2B interacting partners. Our results further implicate Poly [ADP-ribose] polymerases in histone metabolism. Molecular evolutionary analysis, reciprocal affinity purification coupled to mass spectrometry experiments, and indirect immunofluorescence studies using endogenously tagged Spt16Tt (FACT-complex subunit), cNpl1Tt, and PARP6Tt underscore the validity of our approach and offer mechanistic insights. Our results reveal a highly conserved regulatory network for H2A (Hv1)-H2B concerning their nuclear import and assembly into chromatin.