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

Alejandro Saettone,Jean-Philippe Lambert,Kanwal Ashraf,Joanna Derynck,Ronald E Pearlman,Jyoti Garg,Jeffrey Fillingham,Syed Nabeel-Shah

doi:10.1038/s41598-019-56867-0

Abstract

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.

Highlights

The fundamental repeating unit of eukaryotic chromatin, the core nucleosome particle, is comprised of two of each of the core histones H2A, H2B, H3 and H4, and ~147 bp of DNA1
Various metabolic enzymes, including glycolytic enzymes such as pyruvate kinase M2 isoform (PKM2), 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase 4 (PFKFB4), fructose-1,6-bisphosphatase 1 (FBP1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and tricarboxylic acid (TCA) cycle enzymes such as α-ketoglutarate dehydrogenase (α-KGDH) and fumarase as well as enzymes involved in nucleotide synthesis such as inosine 5′-monophosphate dehydrogenase (IMPDH) and GMP synthase (GMPS), have been shown to localize to the nucleus where they may participate in chromatin regulation by modifying the histones and/or supplying metabolites necessary for histone or chromatin modifying enzymes[12]
The expression of endogenously tagged HHO1-FZZ was confirmed by Western blotting analysis in whole cell extracts (WCEs) prepared either from HHO1-FZZ expressing cells or untagged wild-type Tetrahymena (Fig. 1B, right)

Summary

Introduction

The fundamental repeating unit of eukaryotic chromatin, the core nucleosome particle, is comprised of two of each of the core histones H2A, H2B, H3 and H4, and ~147 bp of DNA1. Core histone H3.1 (or H3.2) is assembled onto chromatin only during S phase in a DNA replication dependent manner (RD) by a heterotrimeric CAF1 complex, whereas the variant H3.3 is deposited throughout the cell cycle in a replication independent (RI) fashion by the HIRA histone chaperone[14,15,16,17]. It was reported that human linker histones associated with functionally diverse proteins including RNA-binding proteins, transcriptional regulators, as well as ribosomal proteins[20] In addition to their roles in core histone metabolism, several proteins including nucleosome assembly protein 1 (Nap1) and NASP, are thought to function as H1 chaperones[2,19,21,22,23], mechanistic details remain largely unknown[24]. Starvation in Tetrahymena is a physiological state that is known to induce numerous behavioral, phenotypic and molecular alterations making cells competent to embark on sexual development[32,33]

Methods

Results

Conclusion