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Mol Biol Evol. 2019 May 1;36(5):1037-1055. doi: 10.1093/molbev/msz039.

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

Author information

1
Department of Biology, York University, Toronto, ON, Canada.
2
Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada.
3
Donnelly Centre, University of Toronto, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
4
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
5
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
6
Department of Molecular Medicine and Cancer Research Centre, Université Laval, Québec, QC, Canada.
7
CHU de Québec Research Center, CHUL, Québec, QC, Canada.

Abstract

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 β6 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.

KEYWORDS:

FACT; histone; histone H2A; histone H2B; histone chaperone

PMID:
30796450
PMCID:
PMC6502085
DOI:
10.1093/molbev/msz039
[Indexed for MEDLINE]
Free PMC Article

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