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. 2019 Sep 19;47(16):8399-8409.
doi: 10.1093/nar/gkz547.

A Novel Histone H4 Variant H4G Regulates rDNA Transcription in Breast Cancer

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Free PMC article

A Novel Histone H4 Variant H4G Regulates rDNA Transcription in Breast Cancer

Mengping Long et al. Nucleic Acids Res. .
Free PMC article

Abstract

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

Figures

Figure 1.
Figure 1.
H4G expression in breast cancer patient tissues. (A) Amino acids sequence alignment between primate histone H4G and canonical H4 in primates. Unique amino acids to H4G in comparison with canonical human H4 are highlighted in yellow. (B) Relative H4G expression (mean ± SEM) obtained by quantitative real-time PCR in breast, colon, kidney, liver, lung, ovary, prostate and thyroid normal versus corresponding tumor tissues using the Cancer Survey qPCR panel (#CSRT101, Origene Technologies). *P < 0.05 and **P < 0.01 (Student's t-test). (C) The relative H4G expression obtained from quantitative real-time PCR in breast cancer patient breast tissues and normal breast tissues (#BCRT302, Origene Technologies). The black lines represent the mean expression level of h4g. (D) The relative H4G expression in breast cancer stages (#BCRT302, Origene Technologies). The black lines represent the mean expression level of h4g.
Figure 2.
Figure 2.
H4G interferes with the formation of nucleosome. (A) SDS-PAGE of the canonical histone mixture and the H4G mixture for nucleosome loading. Asterisk (*) represents the GST-H4 and GST-H4G proteins. (B) MNase treatment assay for the GST-H4 and the GST-H4G nucleosomes without DNA extraction. (C) MNase treatment assay for the GST-H4 and the GST-H4G nucleosomes with DNA extraction. 147 represents 147 bp PCR amplified DNA. (D) SDS-PAGE of reconstituted nucleoprotein complexes.
Figure 3.
Figure 3.
H4G localizes to the nucleolar in breast cancer cells. The cellular localization of H4 and H4G in MCF7, LCC1 and LCC2 cells. Each image is representative of MCF7 H4: N = 192 (100%) and MCF7 H4G: N = 110 (75%). The scale bars represent 10 μm.
Figure 4.
Figure 4.
The α-helix 3 of H4G is important for the nucleolar localization. (AH) The cellular localizations of the H4G/H4 hybrid constructs in MCF7 cells. Each image is representative of (A) H4sN: N = 131 (99%), (B) H4GsN: N = 160 (88%), (C) H4sC: N = 105 (100%), (D) H4GsN+C: N = 123 (100%), (E) H4sα1: N = 207 (97%), (F) H4sα2: N = 208 (96%), (G) H4sα3: N = 126 (31%) and (H) H4Gsα3: N = 132 (100%). The scale bars represent 10 μm.
Figure 5.
Figure 5.
H4G strongly interacts with NPM1 and this interaction is mediated by α-helix 3 domain. Immunoprecipitation using FLAG-proteins to identify the interaction of H4G with the nucleolar histone chaperone NPM1.
Figure 6.
Figure 6.
H4G is involved in rRNA transcription. (A) Comparison of rRNA amounts among WT and H4G knockout (H4GKO) MCF7 cell lines. Total RNA was normalized by GAPDH mRNA and loaded onto agarose gels. Error bars represent SD in triplicate experiments. *P < 0.05 and **P < 0.01 (Student's t-test). (B) The relative protein synthesis rate among WT and H4GKO MCF7 cell lines. Error bars represent SD. **P < 0.01. (C) Cell-cycle histograms acquired by flow cytometry and quantification of the subpopulation fraction of the histogram among WT and H4GKOs MCF7 cells. FACS plots and data are representative of at least three separate experiments. (D) The percentage of S phase cells among WT and H4GKOs MCF7 cells. Error bars represent SD. *P < 0.05, ***P < 0.001 (Student's t-test). (E) Representative photographs of the tumors from mouse xenograft model obtained with WT and H4GKO MCF7 cells. The scale bars represent 1 mm. (F) Tumor weight of the MCF7 cell and H4GKO MCF7 cells mouse xenograft models. The black lines represent the mean of tumor weight.

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