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, 10 (1), 168

Nucleus-specific Linker Histones Hho1 and Mlh1 Form Distinct Protein Interactions During Growth, Starvation and Development in Tetrahymena Thermophila

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Nucleus-specific Linker Histones Hho1 and Mlh1 Form Distinct Protein Interactions During Growth, Starvation and Development in Tetrahymena Thermophila

Syed Nabeel-Shah et al. Sci Rep.

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Hho1-FZZ expression and localization in the MAC. (A) Predicted secondary structure of Tetrahymena MAC-specific linker histone Hho1. Hho1 protein is mostly composed of disordered regions. (B) Left- Schematic representation of epitope tagging strategy for the MAC locus. Right- Expression analysis of endogenously tagged Hho1-FZZ in comparison to the untagged controls by Western blotting in whole cell extracts (WCEs). Top panel was probed with anti-FLAG in order to detect Hho1-FZZ and the bottom panel was probed with anti-Actin as a loading control. (C) Left- Western blotting analysis using Hho1-FZZ WCEs to examine its expression across the indicated stages of the Tetrahymena life cycle. VEG-vegetative growth; STV-starvation; mating-conjugating Tetrahymena cells. Top panel was probed with anti-FLAG antibody to detect Hho1-FZZ and the bottom panel was probed with anti-Brg1 as a loading control. Right- HHo1-FZZ localizes to the MAC only and not to the MIC during vegetative growth, starvation and conjugation. Hho1-FZZ cells of mating type VII were mated with the untagged cells of mating type II. Nuclear events are depicted as cartoons. DAPI was used to stain the nuclei. The images were processed using ImageJ (version 1.50i) (https://imagej.nih.gov/ij/).
Figure 2
Figure 2
AP-MS analysis of Hho1-FZZ in three different physiological states of the Tetrahymena life cycle. Top: Western blotting analysis indicating recovery of the affinity purified Hho1-FZZ bait protein. The top panel represents the vegetative samples and the bottom panel shows the recovery of the bait from conjugation samples. The blots were probed with anti-FLAG antibody. No signal was detected in samples from the untagged control cells. Bottom: Network illustration of the Hho1-FZZ co-purifying proteins at statistical threshold of FDR ≤ 1% in three different stages of the Tetrahymena life cycle; vegetative growth, starvation (24 hours) and conjugation (6 hours post mixing of the cells). Node color key is provided.
Figure 3
Figure 3
Mlh1 and HMG-box proteins in Tetrahymena. (A) Cartoon diagram shows the predicted structure of the Tetrahymena Mlh1 HMG-box 1 in rainbow color. The N- and C-termini are indicated. (B) Domain architecture of the HMG-family proteins identified in Tetrahymena. Domain legends are provided in the box. (C) Neighbour-joining phylogeny of HMG-box amino acid sequences from the identified HMG-family proteins in Tetrahymena. 1000 bootstrap replicates were performed to test the reliability of the resulting tree. All the nodes depicted had >70 bootstrap values. Node length does not indicate the genetic distance. (D) Heatmap represents the expression profiles based on hierarchical clustering of the microarray expression values for the HMG-family proteins across different stages of the Tetrahymena life cycle. Z-scores were calculated across each row to examine the differential expression across different stages. L1-LH: vegetative growth, S0-24: starvation for 24 hours, and C: conjugation (0–18 hours post mixing the cells of different mating types).
Figure 4
Figure 4
Mlh1-FZZ exclusively localizes to the MIC. (A) Left: Schematic representation of proteolytic cleavage of full length Mlh1. The predicted product size and molecular weights are indicated. The position and size of the FZZ tag is indicated in blue. Proteolysis of the full-length Mlh1-FZZ results in β-FZZ product. Right: Expression analysis of endogenously tagged Mlh1-FZZ in comparison to the untagged controls by Western blotting in WCEs. The black arrowhead represents the β fragment (~25.5 kDa + FZZ 18 kDa). Anti-FLAG antibody was used to probe the top panel for Mlh1-FZZ detection. The bottom panel was probed with anti-Actin as a loading control. Two separate clones of Mlh1-FZZ were analysed. (B) Mlh1-FZZ localizes to MIC only during vegetative growth, starvation and conjugation. Mlh1-FZZ cells (mating type VII) were mated with untagged cells of mating type II. Nuclear events are illustrated as cartoons. Mlh1-FZZ signal was observed only in the MICs of the FZZ tagged cells and not in the untagged controls. The signal observed in both mating pairs (Mlh1-FZZ and controls) at the anlagen stage indicates mixing of cellular contents between the pairing cells. DAPI was used to stain the nuclei. The images were processed using ImageJ (version 1.50i) (https://imagej.nih.gov/ij/).
Figure 5
Figure 5
Mlh1-FZZ protein-protein interaction network. (A) A Network illustration of the Mlh1-FZZ co-purifying proteins from three different stages of the Tetrahymena life cycle (statistical threshold of FDR ≤ 1%). The node color legend is provided. (B) Heat map of microarray expression values for Mlh1-FZZ co-purifying proteins in the three different stages of the Tetrahymena life cycle. Z-scores were calculated across the rows for each protein to examine its differential expression across growth, starvation and various developmental stages. L1-LH: vegetative growth phase, S0-24: starvation for 24 hours, and C: conjugation where 0–18 denotes hours post mixing the different mating types. V, S and C in parenthesis after each prey protein name indicate vegetative, starvation and conjugation-specific interactions.

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