Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were establish...Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection 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. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. 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 region of 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. Conclusions 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 expres-sion.展开更多
Cells need to appropriately balance transcriptional stability and adaptability in order to maintain their identities while responding robustly to various stimuli. Eukaryotic cells use an elegant "epigenetic"...Cells need to appropriately balance transcriptional stability and adaptability in order to maintain their identities while responding robustly to various stimuli. Eukaryotic cells use an elegant "epigenetic" system to achieve this functionality. "Epigenetics" is referred to as heritable information beyond the DNA sequence, including histone and DNA modifications, nc RNAs and other chromatin-related components. Here, we review the mechanisms of the epigenetic inheritance of a repressive chromatin state,with an emphasis on recent progress in the field. We emphasize that(i) epigenetic information is inherited in a relatively stable but imprecise fashion;(ii) multiple cis and trans factors are involved in the maintenance of epigenetic information during mitosis; and(iii) the maintenance of a repressive epigenetic state requires both recruitment and self-reinforcement mechanisms.These mechanisms crosstalk with each other and form interconnected feedback loops to shape a stable epigenetic system while maintaining certain degrees of flexibility.展开更多
Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intr...Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vlTSs) are also localized at the centro- meric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere se- quences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluo- rescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved te- lomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.展开更多
基金Supported by National Natural Science Foundation of China (30721063)National Basic Research Program of China (973 Program) (2005CB522402, 2006CB910403)+1 种基金National Laboratory of Medical Molecular Biology grant (2060204)Beijing municipal government grant (YB20081002301)
文摘Objective To study the regulatory roles of SIRT1 on EZH2 expression and the further ef-fects on EZH2's repression of target gene expression. Methods The stable SIRT1 RNAi and Control RNAi HeLa cells were established by in-fection 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. Results Western blot results showed that EZH2 protein level increased upon SIRT1 de-pletion. 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 region of 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. Conclusions 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 expres-sion.
基金supported by the National Natural Science Foundation of China (31761163001, 31701128)
文摘Cells need to appropriately balance transcriptional stability and adaptability in order to maintain their identities while responding robustly to various stimuli. Eukaryotic cells use an elegant "epigenetic" system to achieve this functionality. "Epigenetics" is referred to as heritable information beyond the DNA sequence, including histone and DNA modifications, nc RNAs and other chromatin-related components. Here, we review the mechanisms of the epigenetic inheritance of a repressive chromatin state,with an emphasis on recent progress in the field. We emphasize that(i) epigenetic information is inherited in a relatively stable but imprecise fashion;(ii) multiple cis and trans factors are involved in the maintenance of epigenetic information during mitosis; and(iii) the maintenance of a repressive epigenetic state requires both recruitment and self-reinforcement mechanisms.These mechanisms crosstalk with each other and form interconnected feedback loops to shape a stable epigenetic system while maintaining certain degrees of flexibility.
基金supported by the National Basic Research Program of China(Grant Nos. 2009CB941000 and 2011CBA01002)
文摘Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vlTSs) are also localized at the centro- meric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere se- quences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluo- rescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved te- lomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.