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

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 Spt6 Tt specifically copurify with Hv1. We also identified importin β6 and the T. thermophila ortholog of nucleoplasmin 1 (cNpl1 Tt ) 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 Spt16 Tt (FACT-complex subunit), cNpl1 Tt , and PARP6 Tt 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.

The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation

“…Tetrahymena thermophila, a unicellular eukaryotic model system, provides an excellent system to study the functional conservation or diversification of chromatin-related and gene expression regulatory complexes [20,21]. The Tetrahymena genome encodes an Rpb1 (human POLR2A; yeast RPO21) that lacks canonical heptad repeats at its CTD, a feature that is shared among all ciliates ( Figures 1A and S1A).…”

Exploring the Histone Acetylation Cycle in the Protozoan Model Tetrahymena thermophila

“…Previously the Allis lab demonstrated that Hv1, like Ibd1, localizes to the MAC of growing cells implicating Hv1 in transcription (Wenkert and Allis, 1984). Tt SWR-C was defined by the intersection of common proteins in Ibd1 and Swc4 AP-MS (Saettone et al, 2018;Ashraf et al, 2019) with clear orthologs identified for Swr1, Yaf9, Rvb1, RvB2, Swc2, Swc4, and Swc5. AP-MS of Hv1 co-purified Swr1, Swc2, Arp5, and Rvb1 (Ashraf et al, 2019), consistent with Tt SWR-C function in the deposition of Hv1.…”

“…Tt SWR-C was defined by the intersection of common proteins in Ibd1 and Swc4 AP-MS (Saettone et al, 2018;Ashraf et al, 2019) with clear orthologs identified for Swr1, Yaf9, Rvb1, RvB2, Swc2, Swc4, and Swc5. AP-MS of Hv1 co-purified Swr1, Swc2, Arp5, and Rvb1 (Ashraf et al, 2019), consistent with Tt SWR-C function in the deposition of Hv1. In yeast, the NuA4 HAT acetylates H4 which recruits SWR via the BRD-containing Bdf1 subunit to deposit Htz1 (Durant and Pugh, 2007;Altaf et al, 2010).…”

“…Group IIB proteins also include BroP6, BroP2, and BroP4, that are similar to BroP5/Ibd1 in that they are small (∼400 aa), with a single N-terminal BRD and no other recognizable domains. BroP6 in particular is highly similar to Ibd1/BroP5 in amino acid sequence and along with Ibd1 co-purifies in Hv1 AP-MS (Ashraf et al, 2019). INO80-C is a multi-subunit ATP-dependent CR complex (Poli et al, 2017) that possesses a histone-exchange activity that swaps nucleosomal H2A.Z/H2B with H2A/H2B (Papamichos-Chronakis et al, 2011) (in essence, the opposite of SWR-C).…”

“…Both the MAC ( Eisen et al, 2006 ) and MIC ( Hamilton et al, 2016 ) genomes have been sequenced and annotated ( Stover et al, 2006 ). A well-developed functional proteomic pipeline exists for the study of epigenetic regulators in particular for a ffinity p urification coupled to m ass s pectrometry (AP-MS) to effectively solubilize native (i.e., un-crosslinked) protein complexes both bound and unbound to chromatin ( Xiong et al, 2011 ; Garg et al, 2013 , 2019 ; Saettone et al, 2018 , 2019a ; Ashraf et al, 2019 ; Nabeel-Shah et al, 2020 ). A critical feature of this approach is the expression of the epitope tagged proteins at levels closely approximating that of the endogenous protein.…”