Epigenetic regulation of ferroptosis by H2B monoubiquitination and p53
2019; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês
10.15252/embr.201847563
ISSN1469-3178
AutoresYufei Wang, Yang Lü, Xiaojun Zhang, Wen Cui, Yan Liu, Qinru Sun, Qing He, Shiyan Zhao, Guoan Zhang, Yequan Wang, Su Chen,
Tópico(s)RNA modifications and cancer
ResumoArticle22 May 2019free access Source DataTransparent process Epigenetic regulation of ferroptosis by H2B monoubiquitination and p53 Yufei Wang Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Lu Yang Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Xiaojun Zhang Department of Science and Education, People's Hospital of Zunhua, Tangshan, Hebei, China Search for more papers by this author Wen Cui Corresponding Author [email protected] School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Yanping Liu Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Qin-Ru Sun Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Qing He Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Shiyan Zhao Community Health Service Center of Yaoqiang, Jinan, Shandong, China Search for more papers by this author Guo-An Zhang School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Yequan Wang School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Su Chen Corresponding Author [email protected] orcid.org/0000-0001-8804-8989 Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Department of Science and Education, People's Hospital of Zunhua, Tangshan, Hebei, China School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Yufei Wang Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Lu Yang Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Xiaojun Zhang Department of Science and Education, People's Hospital of Zunhua, Tangshan, Hebei, China Search for more papers by this author Wen Cui Corresponding Author [email protected] School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Yanping Liu Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Qin-Ru Sun Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Qing He Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Search for more papers by this author Shiyan Zhao Community Health Service Center of Yaoqiang, Jinan, Shandong, China Search for more papers by this author Guo-An Zhang School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Yequan Wang School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Su Chen Corresponding Author [email protected] orcid.org/0000-0001-8804-8989 Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China Department of Science and Education, People's Hospital of Zunhua, Tangshan, Hebei, China School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China Search for more papers by this author Author Information Yufei Wang1,‡, Lu Yang1,‡, Xiaojun Zhang2,‡, Wen Cui *,3, Yanping Liu1, Qin-Ru Sun1, Qing He1, Shiyan Zhao4, Guo-An Zhang3, Yequan Wang3 and Su Chen *,1,2,3 1Laboratory of Molecular and Cellular Biology, School of Forensic Sciences, Center for Translational Medicine at The First Affiliated Hospital, Xi'an Jiao Tong University Health Science Center, Xi'an, Shaanxi, China 2Department of Science and Education, People's Hospital of Zunhua, Tangshan, Hebei, China 3School of Forensic Sciences and Laboratory Medicine, Jining Medical University, Jining, Shandong, China 4Community Health Service Center of Yaoqiang, Jinan, Shandong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 0537 3616219; E-mail: [email protected] *Corresponding author. Tel: +86 029 82655299; E-mail: [email protected] EMBO Rep (2019)20:e47563https://doi.org/10.15252/embr.201847563 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Monoubiquitination of histone H2B on lysine 120 (H2Bub1) is an epigenetic mark generally associated with transcriptional activation, yet the global functions of H2Bub1 remain poorly understood. Ferroptosis is a form of non-apoptotic cell death characterized by the iron-dependent overproduction of lipid hydroperoxides, which can be inhibited by the antioxidant activity of the solute carrier family member 11 (SLC7A11/xCT), a component of the cystine/glutamate antiporter. Whether nuclear events participate in the regulation of ferroptosis is largely unknown. Here, we show that the levels of H2Bub1 are decreased during erastin-induced ferroptosis and that loss of H2Bub1 increases the cellular sensitivity to ferroptosis. H2Bub1 epigenetically activates the expression of SLC7A11. Additionally, we show that the tumor suppressor p53 negatively regulates H2Bub1 levels independently of p53's transcription factor activity by promoting the nuclear translocation of the deubiquitinase USP7. Moreover, our studies reveal that p53 decreases H2Bub1 occupancy on the SLC7A11 gene regulatory region and represses the expression of SLC7A11 during erastin treatment. These data not only suggest a noncanonical role of p53 in chromatin regulation but also link p53 to ferroptosis via an H2Bub1-mediated epigenetic pathway. Overall, our work uncovers a previously unappreciated epigenetic mechanism for the regulation of ferroptosis. Synopsis This study uncovers a previously unknown epigenetic regulatory mechanism for the control of ferroptosis by p53-USP7-H2Bub1 axis. H2Bub1 is a novel epigenetic regulator of ferroptosis. p53 regulates H2Bub1 independent of its transcription factor activity by recruiting the H2Bub1 deubiquitinase USP7 to chromatin. p53-USP7 interplay reduces the occupancy of H2Bub1 on the SLC7A11 regulatory region, resulting in transcriptional repression and increased sensitivity to ferroptosis induction. These results not only suggest a novel role of H2Bub1 in the regulation of ferroptosis but also reveal a relative direct role of p53 in the regulation of chromatin events. Introduction Cell death plays crucial roles in multiple biological processes, such as the control of normal development, the maintenance of homeostasis in multicellular organisms, and the regulation of uncontrolled proliferative or degenerative diseases 1-3. Apoptosis is the most deeply investigated form of cell death, while other types of non-apoptotic cell death (e.g., necroptosis or pyroptosis) have also been identified. Ferroptosis is a recently discovered type of non-apoptotic cell death 4-7. The execution of ferroptosis closely involves the ion-dependent accumulation of lipid reactive oxygen species (ROS) instead of cytosolic ROS. The small molecule erastin has been recognized as specifically inducing ferroptosis by disrupting cellular lipid redox homeostasis. Detailed studies indicated that erastin inhibits the activity of the cystine/glutamate antiporter (system ), further leading to a decrease in cystine uptake. This reduction in cystine eventually results in a depletion of glutathione (GSH), which is the major antioxidant in cells 8, 9. Reduced GSH is utilized by glutathione peroxidase 4 (GPX4) to reduce lipid hydroperoxides and therefore protects cells against ferroptosis. The inactivation of GPX4 induces ferroptosis even in cells with normal cystine and GSH contents 10. SLC7A11 is a key component of system , which is required for cystine uptake 11, 12. Consistently, depletion of SLC7A11 results in significant upregulation of erastin-induced ferroptosis. In addition, ferroptosis is morphologically distinct from other types of programmed cell death. For instance, ferroptotic cells show abnormal mitochondrial morphology with smaller size and increased membrane density, which has been recognized as the lone typical characteristic of ferroptosis in terms of morphology 4, 13. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a key epigenetic modification in the regulation of gene transcription and chromatin organization. For example, H2Bub1 is usually believed to be an active marker for gene transcription and results in an open chromatin context to facilitate gene transcription 14-17. H2Bub1 is catalyzed by the E2 ubiquitin-conjugating enzyme RAD6 (UBE2A/B) and E3 ubiquitin-protein ligase BRE1 (RNF20/40), and the regulation of H2Bub1 by RAD6/BRE1 is conserved from yeast to human 18-21. In addition to the establishing enzymes, multiple deubiquitinases have also been identified, including USP7 22-25, USP12 26, USP22 27, USP44 28, USP46 26, and USP49 29. An increasing number of studies have indicated that H2Bub1 plays significant roles in various biological processes. For example, other researchers and our group have revealed the critical roles of H2Bub1 in the control of embryonic stem cell (ESC) differentiation. Levels of H2Bub1 are increased significantly during ESC differentiation, and this upregulation of H2Bub1 is required for efficient ESC differentiation. Mechanistic studies have revealed that the role of H2Bub1 in the regulation of ESC differentiation is mainly achieved through epigenetically regulating the expression of differentiation-related genes or a group of long genes 28, 30, 31. Moreover, H2Bub1 has also been reported to be closely involved in cancer. Most studies have indicated that the levels of H2Bub1 are decreased in cancer, and loss of H2Bub1 promotes cancer metastasis 32, 33, although controversial results have been reported 34. In addition, our group recently suggested that H2Bub1 is also a critical epigenetic marker for the regulation of autophagy. H2Bub1 levels are strikingly decreased during starvation-induced autophagy, and loss of H2Bub1 results in autophagosome formation and the activation of autophagy 35, 36. p53 is the most investigated tumor suppressor and is mutated in more than 50% of human cancers 37-39. p53 is a sequence-specific DNA-binding protein that functions as a transcription factor 40, 41. It is well known that p53 plays critical roles in apoptosis, cell cycle regulation, senescence, DNA repair, and other cellular events 42. A recent study indicated that p53 also plays a crucial role in the control of ferroptosis by downregulating the expression of SLC7A11 13. However, how p53 negatively regulates SLC7A11 expression is still unclear, since p53 is a traditional transcription factor that usually activates gene expression. In addition, the chromatin-regulating function of p53 has recently become a focus. p53 “gain-of-function” mutants can affect histone methylation and acetylation by regulating the expression of histone methyltransferases (e.g., MLL1 and MLL2) and histone acetyltransferase (e.g., MOZ) 43. Nevertheless, the role of p53 in controlling histone methylation and acetylation is still achieved through transcriptionally regulating the expression of the related enzymes. In this study, we demonstrate that H2Bub1, as an epigenetic modification, plays an essential role in the regulation of ferroptosis. In addition, we identified that p53 is a novel regulator of H2Bub1, likely independent of its transcription factor activity, further supporting the status of p53 as a significant chromatin regulator 43. Most importantly, this study also links p53 to ferroptosis by the H2Bub1-mediated epigenetic pathway. Results Loss of H2Bub1 significantly sensitizes cells to erastin-induced ferroptosis To determine whether epigenetic modifications regulate ferroptosis, we first performed Western blot screening with various antibodies against distinct histone modifications upon erastin (a specific ferroptosis inducer) stimulation, a standard method to induce ferroptosis 4, 13. Intriguingly, we found that the levels of H2Bub1 are strikingly decreased after erastin treatment, suggesting that H2Bub1 may be involved in the regulation of ferroptosis (Fig 1A). To further determine the role of H2Bub1 in the control of ferroptosis, we next tested the effect of loss of H2Bub1 on ferroptosis. In addition to knockdown of RNF20 expression, we and others previously indicated that overexpression of the K120R mutant of H2B (H2BK120R) can also downregulate the levels of endogenous H2Bub1 making a useful way to examine the roles of H2Bub1 44, 45. Therefore, we inhibited endogenous H2Bub1 by knocking down the RNF20 expression and overexpressing of H2BK120R mutant (Fig EV1A). Surprisingly, we observed that loss of H2Bub1 with RNF20-specific siRNA or H2BK120R mutant significantly sensitizes cells to erastin-induced cell death (Figs 1B and EV1B). As mentioned above, lipid ROS accumulation is a hallmark of ferroptosis 4. We therefore examined the intracellular lipid ROS levels in normal and H2Bub1-depleted cells upon erastin treatment. Consistent with its effect on cell death, the loss of H2Bub1 obviously increased erastin-induced lipid ROS levels (Figs 1C, and EV1C and D). Because the alteration of mitochondrial morphology is the lone characteristic feature of ferroptosis compared to other forms of cell death 4, 13, we performed a transmission electron microscopy assay to detect mitochondrial morphology. Consistent with the ferroptotic morphology 4, 13, cells depleted of H2Bub1 and treated with erastin show very serious defects in mitochondrial morphology, including smaller size and increased membrane density, while the nuclear membrane is intact, and no DNA fragmentation is observed (Fig 1D). To further confirm the mode of H2Bub1-regulated cell death upon erastin stimulation, we treated cells with ferrostatin-1 (ferr-1), a specific inhibitor of ferroptosis 4, 13. Notably, ferr-1 almost completely rescued the cell death induced by the loss of H2Bub1 upon erastin treatment (Fig 1E). In contrast, inhibitors of other forms of cell death, including ZVAD-FMK (apoptosis), necrostatin-1 (necroptosis), and 3-MA (autophagy), all failed to suppress erastin-induced cell death in H2Bub1-depleted cells (Fig EV1E and F). Together, these data demonstrate that the epigenetic marker H2Bub1 is likely a novel negative regulator of ferroptosis. Figure 1. Loss of H2Bub1 promotes ferroptosis Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times. H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (−) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated as in (B). H1299 cells treated as in (B) were subjected to transmission electron microscopy. Representative images are shown. Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean ± s.d., n = 3 independent repeats. Two-tailed unpaired Student's t-test was performed. *P < 0.05, **P < 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of H2Bub1 sensitizes cells to erastin-induced cell death A. Western blot for H1299 cells transfected as indicated. B. H1299 cells transfected with a control siRNA (siCont.) or a RNF20-specific siRNA (siRNF20) were treated with erastin with different dosage for 24 h or with 12 μM for different time points as indicated. Cell death was then examined by PI staining coupled with flow cytometry assay. C, D. H1299 cells transfected as indicated for 24 h were treated with 12 μM erastin (+) or untreated (−) for another 24 h. Cells were harvested and subjected to flow cytometry analysis by staining with C11-BODIPY to detect lipid ROS levels. E, F. H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20), and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin for 24 h together with specific cell death inhibitors (+) or untreated (−). Representative phase-contrast images were recorded (magnification, ×20) (E), and the surviving cells were counted (F). Data information: Bars and error bars are mean ± s.d., n = 3 independent repeats. Download figure Download PowerPoint H2Bub1 regulates the expression of SLC7A11 and a group of ion-binding genes that function in multiple metabolism-related processes As previously mentioned, SLC7A11, which encodes a component of the cystine/glutamate antiporter, system , is a critical protein in the control of ferroptosis 4, 13. Therefore, we tested whether H2Bub1 affects SLC7A11 expression. To our surprise, we found that loss of H2Bub1 significantly downregulates both the mRNA and protein levels of SLC7A11 (Fig 2A). Moreover, our chromatin immunoprecipitation (ChIP) analysis indicates that H2Bub1 is enriched in the gene regulatory region of the SLC7A11 gene (Fig 2B, left), and most importantly, erastin treatment abolishes the occupancy of H2Bub1 on SLC7A11 (Fig 2B, right), suggesting that SLC7A11 may represent a novel downstream target gene of H2Bub1. As above mentioned, the uptake of extracellular cystine is mediated by SLC7A11, and cystine is a major precursor for GSH biosynthesis. GSH is the primary cellular antioxidant and protects cells from ferroptosis 8-12. We therefore tested the intracellular GSH levels to indicate the activities of SLC7A11. Consistently, we observed that loss of H2Bub1 decreased the intracellular GSH levels, suggesting an impaired SLC7A11 activity in H2Bub1-depleted cells (Fig 2C). We next examined whether SLC7A11 is essential for the sensitization of cells to erastin-induced ferroptosis by loss of H2Bub1. As expected, SCL7A11 overexpression almost significantly rescued the ferroptosis induced by the loss of H2Bub1 upon erastin stimulation, suggesting that SLC7A11 plays a major role in mediating the loss of H2Bub1-sensitized ferroptosis (Fig 2D and E). Figure 2. Identification of SLC7A11 as a target of H2Bub1 qRT–PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (−) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). Surviving cells from the assay shown in (D) were counted. GO analysis with the genes downregulated in H2BK120R (black) or RNF20-specific siRNA (siRNF20) (red) transfected 293T cells by employing a previously reported microarray data 44. Affected metal ion-binding genes in (F) were selected and subjected to cluster analysis. Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells. Labile iron levels examined in (H) were quantified. Data information: Bars and error bars are mean ± s.d., n = 3 independent repeats. Two-tailed unpaired Student's t-test was performed. *P < 0.05, **P < 0.01. Download figure Download PowerPoint In addition, we previously performed a microarray analysis to predict the global biological roles of H2Bub1 with H2Bub1-depleted cells 44. A GO analysis was performed with these reported data, and intriguingly, we found that a group of genes that encode metal ion-binding proteins were significantly affected by the loss of H2Bub1 (Fig 2F), and these genes were downregulated after H2Bub1 depletion (Figs 2G, and EV2A and B). We therefore suspected that the decreased expression of these genes after loss of H2Bub1 may result in an increase in metal ion levels that may include iron, providing a novel possible mechanism for the sensitization of cells to erastin-induced ferroptosis by loss of H2Bub1. Therefore, we measured the labile iron levels in both control and H2Bub1-depleted H1299 cells. Consistent with our speculation, the results show that loss of H2Bub1 significantly increases the labile iron levels in human cells (Fig 2H and I). In addition, a STRING analysis suggested that these affected metal ion-binding genes are mainly enriched in various metabolism-related processes, including redox-related events (e.g., ADAMTS5, CYP51A1, and ZNF341) and multiple macromolecule metabolic processes (e.g., BCL11B, MGAT4A, CHSY3, and LMO3; Fig EV2C), which are tightly related to the regulation of cellular redox balance. Click here to expand this figure. Figure EV2. H2Bub1 regulates the expression of a group of ion-binding proteins mRNA levels of H2Bub1-affected metal ion-binding genes in H1299 cells transfected with wild-type H2B (H2BWT) and K120R-mutated H2B (H2BK120R) were determined by RT–PCR analysis. The mRNA levels of the same genes shown in (A) were tested by RT–PCR analysis in H1299 cells transfected with a control siRNA (siCont.) and an RNF20-specific siRNA (siRNF20). STRING analysis of the selected metal ion-binding genes indicated that several metabolism-related processes are involved, including multiple redox-related events. Download figure Download PowerPoint p53 negatively regulates H2Bub1 levels Previous studies indicated that p53 controls chromatin methylation and acetylation by transcriptionally regulating the expression of multiple histone methyltransferases and acetyltransferase 43 and that p53 affects ferroptosis by transcriptionally downregulating the expression of SLC7A11 13. Therefore, we wondered whether p53 modulates H2Bub1 levels. To our great surprise, we found that overexpression of p53 reduces H2Bub1 levels strikingly in distinct cell lines (Figs 3A and EV3A), whereas the levels of other histone modifications, including H3K9me3, H3K27me3, H3K36me3, H3K56me1, H4K8ac, and H4K20me3, are not affected (Figs 3B and EV3B). Although H3K4me3 and H3K79me3 are reported to be downstream targets of H2Bub1 46-48, the levels of both H3K4me3 and H3K79me3 were not obviously affected by p53 overexpression (Fig 3A), which is consistent with the view that changes in H2Bub1 are not always coupled with changes in H3K4me3 and H3K79me3 28, 30, 31, 49, 50. Moreover, the results of our immunofluorescence (IF) assay also support the above notion that overexpression of p53 downregulates the levels of H2Bub1 (Fig 3C). Together, these findings suggest that p53 specifically regulates H2Bub1 in human cells. Figure 3. p53 negatively regulates H2Bub1 A, B. Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h. C. Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell. D. Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h. E. 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis. F. SMMC cells were treated with 100 μM etoposide (+) or untreated (−) for 24 h, and Western blot analysis was performed. G. Western blot analysis of A549 cells and H1299 cells. H. H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay. I. Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two “gain-of-function” mutants (p53-R273H and p53-R175H) for 48 h. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Loss of H2Bub1 affects H2Bub1 levels and chromatin compaction A–C. H1299 or Hep3B cells were transfected with a Flag-tagged p53 or an empty control vector (Cont.) for 48 h followed by Western blot analysis. Arrowheads indicate the corresponding band of the indicated proteins. D. Control (siCont.) or p53 depleted (sip53) SMMC cells were treated with 100 μM etoposide (+) or untreated (−) for 24 h as indicated, and Western blot analysis was performed. E. Relative quantification of 1N–3N bands specifically corresponding to the data in Fig 3H. No replicates were involved in the band quantification. F. MNase digestion analysis for 293T cells transfected with a control siRNA (siCont.) or a p53-specific siRNA. G. 293T cells transfected with a control siRNA (siCont.), a p53-specific siRNA (sip53), or sip53 together with an RNF20-specific siRNA (sip53 + siRNF20) were harvested for the MNase digestion assay. Corresponding relative quantification of 1N–3N bands is shown. No replicates were involved in the band quantification. H. 293T cells transfected with a control siRNA (siCont.), a p53-specific siRNA (sip53) together with a wild-type H2B (H2BWT), or a K120R-mutated H2B (H2BK120R) as indicated for 48 h. Cells were then harvested for the MNase digestion assay. Corresponding relative quantification of 1N–3N bands is shown. No replicates were involved in the band quantification. I. MDA-MB-468 cells infected with a control shRNA (siCont.) and a p53-specific shRNA (sip53, Santa Cruz, #sc-29435-V) were subjected to Western blot analysis. J. MNase digestion analysis for MDA-MB-468 cells infected with a control shRNA (siCont.) or a p53-specific shRNA. Download figure Download PowerPoint We next wondered how p53 regulates the levels of H2Bub1. Therefore, the levels of all the reported ubiquitinases (e.g., RAD6, RNF20, and RNF40) 20, 21 and deubiquitinases (e.g., USP7, USP12, USP22, USP44, USP46, and USP49) 22-29 were examined upon p53 overexpression. However, no obvious changes were observed in any of the tested enzymes, suggesting that other mechanisms are likely involved (Figs 3D and EV3C). Furthermore, we also performed a p53 knockdown experiment to validate the effect of p53 on H2Bub1. Consistently, the levels of H2Bub1 were increased in p53-depleted cells (Fig 3E). To further confirm p53-H2Bub1 regulation under relative physiological conditions, we tested the relationship between p53 and H2Bub1 in response to treatment with etoposide, a well-known anti-cancer drug that can induce p53 upregulation 51, 52. Intriguingly, the levels of H2Bub1 were significantly decreased, therefore negatively correlating to p53 upregulation upon etoposide treatment (Fig 3F). Moreover, the observed etoposide-induced loss of H2Bub1 can be rescued by further depletion of p53, suggesting that the effect of etoposide on H2Bub1 is p53 dependent (Fig EV3D). More interestingly, we examined the levels of H2Bub1 in a pair of cell lines with (A549) or without p53 (H1299). The levels of H2Bub1 in
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