Histone Chaperone Nrp1 Mutation Affects the Acetylation of H3K56 in Tetrahymena thermophila

Abstract

Histone modification and nucleosome assembly are mainly regulated by various histone-modifying enzymes and chaperones. The roles of histone-modification enzymes have been well analyzed, but the molecular mechanism of histone chaperones in histone modification and nucleosome assembly is incompletely understood. We previously found that the histone chaperone Nrp1 is localized in the micronucleus (MIC) and the macronucleus (MAC) and involved in the chromatin stability and nuclear division of Tetrahymena thermophila. In the present work, we found that truncated C-terminal mutant HA-Nrp1^TrC abnormally localizes in the cytoplasm. The truncated-signal-peptide mutants HA-Nrp1^TrNLS1 and HA-Nrp1^TrNLS2 are localized in the MIC and MAC. Overexpression of Nrp1^TrNLS1 inhibited cellular proliferation and disrupted micronuclear mitosis during the vegetative growth stage. During sexual development, Nrp1^TrNLS1 overexpression led to abnormal bouquet structures and meiosis arrest. Furthermore, Histone H3 was not transported into the nucleus; instead, it formed an abnormal speckled cytoplastic distribution in the Nrp1^TrNLS1 mutants. The acetylation level of H3K56 in the mutants also decreased, leading to significant changes in the transcription of the genome of the Nrp1^TrNLS1 mutants. The histone chaperone Nrp1 regulates the H3 nuclear import and acetylation modification of H3K56 and affects chromatin stability and genome transcription in Tetrahymena.

Keywords: Tetrahymena thermophila; acetylation of H3K56; genome transcription; histone chaperone NRP1; mutation.

Figure 1

Localization of Nrp1 mutants. (A) Schematic representation of Nrp1, Nrp1^TrNLs1, Nrp1^TrNLs2, and Nrp1^Trc truncated mutants. (B) Localization of Nrp1 and Nrp1 mutants during conjugation stage: (a) HA-Nrp1, (b) HA-Nrp1^TrNLS1, (c) HA-Nrp1^TrNLS2, and (d) HA-Nrp1^TrC; (e) WT cell is as negative control.

Figure 2

Proliferation and nuclear development of Nrp1^TrNLS1 mutants. (A) Relative expression level of NRP1^TrNLS1. Total RNA was isolated from cells in the vegetative growth stage. The relative expression level of NRP1 was identified by qRT-PCR. Data were analysed statistically by independent samples t-test, asterisk indicate significant difference (** p < 0.01). (B) Proliferation of the Nrp1^TrNLS1 mutant and WT cells; data represented three independent experiments. The cell concentrations were analyzed by paired-samples t-test, indicated the significant difference (p = 0.02 < 0.05) between Nrp1^TrNLS1 mutant and WT cells. (C) Analysis of cellular apoptosis by flow cytometry. (D) Representative images of the division of MIC and MAC. (a–c) WT as the negative control, DNA was stained with DAPI. (d–i) Lost MIC and abnormally divided MAC were observed in the Nrp1^TrNLS1 mutants. Scale bar, 10 µm. (E) M, trans 2 K plus DNA marker. MIC, specific sequences were amplified by PCR with 10 sets of primers. Primers Ⅰ was designed for JMJ1, primers Ⅱ–Ⅺ were designed for five different chromosomes in MIC. JMJ1 was used as the internal control.

Figure 3

Overexpression of Nrp1^TrNLS1 affects H3 nuclear import. Indirect immunofluorescence localization of H3. The WT (a–c) and Nrp1^TrNLS1 (d–f) mutant cells were induced with 0.3 µg/mL Cd²⁺ for 24, 48, and 96 h. The primary antibody was histone H3 rabbit polyclonal antibody, and the secondary antibody was FITC-conjugated goat anti-rabbit. Arrows indicate MIC, arrowheads indicate MAC. Scale bar, 10 µm.

Figure 4

Nrp1 mutation leads to reductions in H3K56ac and H4K5ac. (A) Indirect immunofluorescence localization of H3K56ac and H4K5ac. The cells were induced with 0.3 µg/mL Cd²⁺ for 24, 48, and 96 h. The primary antibody was histone H3K56ac or H4K5ac rabbit polyclonal antibody, and the secondary antibody was FITC-conjugated goat anti-rabbit. White arrows indicate MIC, while white arrowheads indicate MAC. Scale bar, 10 µm. (B) Western blot analysis of H3K4me3, H3K56ac, and H4K5ac. Histone extracts prepared from WT and Nrp1^TrNLS1 cells at the vegetative growth stage were induced with 0.3 µg/mL Cd²⁺ for 96 h. The extracted histone samples were separated by 15% SDS–PAGE. The gels were transferred to polyvinylidene difluoride membranes and probed by using anti-H3K4me3, H3K56ac, and H4K5ac antibodies. (C) The intensities of H3K4me3, H3K56ac, and H4K5ac were obtained by ImageJ software. The fluorescence of H3K4me3, H3K56ac, and H4K5ac in WT cells was arbitrarily set as 1, and the Nrp1^TrNLS1 mutation fluorescence was normalized to signal from WT cells.

Figure 5

Micronuclear bouquet formation is affected in the Nrp1^TrNLS1 mutant. (A) Microscopic analysis of changes in the MIC structure during meiosis. In the Nrp1^TrNLS1 mutant, MICs failed to develop to the crescent stage. DNA was stained with DAPI; scale bar, 10 µm. (B) Development of the nucleus during sexual development. Percentage of different developmental stage cells during the sexual reproduction stage in the Nrp1^TrNLS1 mutant and wild-type cell (n > 300). Cells were fixed at 2, 4, 6, 8, 10, and 24 h after mixing and staining with DAPI. (C) Spindle microtubule assembly was analyzed by indirect immunofluorescence localization. White arrowheads indicate spindle microtubules. The experiments were repeated thrice. (a,b) WT, (c,d) Nrp1^TrNLS1 mutant. Scale bar, 10 µm.

Figure 6

Identification of differentially expressed genes in Nrp1^TrNLS1 mutant. (A) Heatmap of DEGs in WT and Nrp1^TrNLS1 cells. DEGs with a |log2FC| > 1 are indicated in red, and DEGs with a |log2FC| < 1 are indicated in green. (B) Volcano plots of DEGs between samples. The threshold q < 0.05 was used to determine the significance of DEGs. Red and green dots represent up- and downregulated genes, respectively, and black dots indicate transcripts that did not change significantly in WT and Nrp1^TrNLS1 cells. (C) KOG enrichment analysis of Nrp1^TrNLS1 and WT cells. (D) KEGG pathway-enrichment analysis of Nrp1^TrNLS1 and WT cells. (E) GO-term enrichment analysis of WT and Nrp1^TrNLS1 cells.

Figure 7

Relative expression level of 10 differentially expressed genes. (A) The expression level of 10 genes was determined by qRT-PCR. (B) Comparison of the log2 of gene expression ratios obtained between the RNA-Seq data and qRT-PCR. The qPCR log₂ value of the expression ratio (y-axis) was plotted against the value from the RNA-Seq (x-axis). R² represents the correlation coefficient between qRT-PCR and RNA-Seq.