-
[show abstract]
[hide abstract]
ABSTRACT: To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.
The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.
Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.
Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.
Chinese Medical Sciences Journal 06/2011; 26(2):77-84.
-
[show abstract]
[hide abstract]
ABSTRACT: A wide variety of nuclear regulators and enzymes are subjected to acetylation of the lysine residue, which regulates different aspects of protein functions. The MYST family histone acetyltransferase, human ortholog of MOF (hMOF), plays critical roles in transcription activation by acetylating nucleosomal H4K16. In this study, we found that hMOF acetylates itself in vitro and in vivo, and the acetylation is restricted to the conserved MYST domain (C2HC zinc finger and HAT), of which the K274 residue is the major autoacetylation site. Furthermore, the class III histone deacetylase SIRT1 was found to interact with the MYST domain of hMOF through the deacetylase catalytic region and deacetylate autoacetylated hMOF. In vitro binding assays showed that non-acetylated hMOF robustly binds to nucleosomes while acetylation decreases the binding ability. In HeLa cells, the recruitment of hMOF to the chromatin increases in response to SIRT1 overexpression and decreases after knockdown of SIRT1. The acetylation mimic mutation K274Q apparently decreases the chromatin recruitment of hMOF as well as the global H4K16Ac level in HeLa cells. Finally, upon SIRT1 knockdown, hMOF recruitment to the gene body region of its target gene HoxA9 decreases, accompanied with decrease of H4K16Ac at the same region and repression of HoxA9 transcription. These results suggest a dynamic interplay between SIRT1 and hMOF in regulating H4K16 acetylation.
Cell Research 04/2011; 21(8):1182-95. · 8.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1.
HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls.
Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay Results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells.
SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.
Chinese Medical Sciences Journal 12/2010; 25(4):199-205.
-
[show abstract]
[hide abstract]
ABSTRACT: Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. By using this approach, specific genetic changes have been created in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. This hypothesis prompted us to systematically investigate SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. We found that the efficiency of SSO-mediated gene repair was elevated by approximately 10-fold in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. We supply evidence suggesting that these increased repair frequencies arise from a thymidine-induced slowdown of replication fork progression. Our studies provide fresh insight into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrate how its efficiency may be reliably and substantially increased.
Proceedings of the National Academy of Sciences 03/2005; 102(7):2508-13. · 9.68 Impact Factor
