Abstract
Background/Aim: CDK inhibitor p16 plays a pivotal role in the induction of cellular senescence and functions as a tumor suppressor. Here, we demonstrate that histone H1.2 is involved in p16 repression. Materials and Methods: Cells were transfected with siRNAs and subjected to quantitative reverse transcription-polymerase chain reaction, immunoblotting and chromatin immunoprecipitation (ChIP) assay. Results: The decrease in H1.2 by oncogenic RAS was associated with increased levels of p16. Depletion of H1.2 selectively increased p16, but not alternative reading frame (ARF) mRNA. ChIP assay showed that H1.2 directly bound to the p16 promoter. Interestingly, silencing YB-1, a component of H1.2 complex, decreased the expression levels of H1.2, resulting in decreased binding of H1.2 on the p16 promoter. Conclusion: These results provide a model in which H1.2 is positively regulated by YB-1 and directly binds to and represses the transcription of p16.
The eukaryotic cell cycle is positively regulated by cyclin and cycle-dependent kinase (CDK) complexes, the activity of which is inhibited by CDK inhibitors (CKIs) including p15, p16, p18, p19, p27 and p57 (1, 2). These CKIs specifically associate and inhibit the enzymatic activity of cyclin-CDK complexes that regulate G1-to-S progression, leading to cell cycle arrest in the G1 phase (3, 4). Among CKIs, p16 plays a pivotal role in the induction of cellular senescence through inhibition of CDK4/6 activity (5, 6). The p16 protein is relatively stable, and its regulation is mainly through transcriptional control (7). The transcription of p16 is tightly repressed during embryogenesis and in young tissues and is activated with age and culture passages, leading to irreversible cell cycle arrest, named replicative senescence (8). This transcription is also activated by oncogenic insults, such as activating RAS mutations, leading to irreversible cell cycle arrest, called premature senescence (9-11). The induction of p16 is required to protect cells from hyperproliferation stimulation. The loss of p16 gene in mice leads to greater propensity to spontaneous cancers (12, 13). In humans, p16 gene is homogenously deleted or silenced in many human cancers with an estimated frequency of approximately 40% (14).
We (15) and Bracken et al. (16) reported that polycomb repression complex 1 (PRC1) and PRC2 bind to and repress the p16 locus though histone H3 lysine 27-trimethylation. A long noncoding RNA, ANRIL binds to PRC1 and PRC2 and brings them on the p16 locus (17, 18).
Histone H1 acts as a linker of nucleosomes and influences chromatin structure, leading to repression of gene expression (19-21). Histone H1 has at least eight subtypes including H1.1-H1.5, H1o, H1t and H1oo. The expression pattern of each subtype is dependent on tissue, stage of development, and cell condition (22-24). Among these subtypes, H1.2 forms complexes with the transcription factor YB-1 to repress p53-mediated transcription (25). We previously reported that YB-1 directly binds to and represses p16 transcription, leading to the repression of cellular senescence (26). In this study, we investigated the involvement of H1.2 in the repression of p16 transcription.
Materials and Methods
Cell culture and RNA interference. Normal human diploid fetal lung fibroblasts WI38 were obtained from the American Type Culture Collection (Frederick, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum (Gibco, Grand Island, NY, USA). WI38 cells were infected with retroviruses harbouring H-RASG12V. Retrovirus generation and infection were performed as described previously (15). The infected cells were selected through treatment with 1 μg/ml puromycin for 72 h. WI38 cells were transfected with siRNA oligonucleotides against H1.2 or YB-1 using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The cells were then incubated for 72 h after transfection before analysis. The nucleotide sequences of siRNA oligonucleotides were as follows: human H1.2, 5′-AGAGCGUAGCGGAGUUUCU-3′ with 3′ dTdT overhangs; and human YB-1, 5′-GGUUCCCACCUUACUACAU-3′ with 3′ dTdT overhangs.
Immunoblotting. Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF) and subjected to SDS-PAGE and immunoblotting as described previously (27). The immunoblotting was performed using antibodies against H-RAS (OP23; Calbiochem-Merk, Darmstadt, Germany), H1.2 (ab4086; Abcam, Cambridge, UK), human p16 (ab50282; Abcam), YB-1 (ab12148; Abcam), and α-tubulin (DM1A; Sigma, St. Louis, MO, USA).
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR was performed as described previously (28). The assays were performed in triplicate using an Mx3000P Real-Time Q-PCR System (Agilent Technologies, Santa Clara, CA, USA). The primer sequences were as follows: p16, 5′-CGGTCGGAGGCCGAT CCAG-3′ and 5′-GCGCCGTGGAGCAGCAGCAGCT-3′; ARF, 5′-CCCTCGTGCTGATGCTACTG-3′ and 5′-ACCTGGTCTTCTAGG AAGCGG-3′; H1.2. 5′-ACTTGGTCTCAAGAGCCTGGTG-3′ and 5′-GGCTTCTTAGGTTTGGTTCCGC-3′; and GAPDH, 5′-GCAA ATTCCATGGCACCGT-3′ and 5′-TCGCCCCACTTGATTTTGG-3′.
Chromatin immunoprecipitation (ChIP). A total of 1×106 WI38 cells were fixed with 1% formaldehyde for 10 min at 37°C. ChIP assays were performed by using a ChIP Assay Kit (Upstate, Lake Placid, NY, USA) according to the manufacturer’s protocol. Immunoprecipitation was conducted with antibodies against H1.2 (ab4086; Abcam) or normal rabbit IgG for a negative control. The precipitated DNA was purified using a QIAquick PCR Purification Kit (Qiagen). PCR was performed using Platinum Taq polymerase (Invitrogen). The primer sequences were as follows: p16 locus (a), 5′-GGCATCAGCAA AGTCTGAGC-3′ and 5′-CTGGGAGACAAGAGCGAAAC-3′; and p16 locus (b), 5′-AGGGGAAGGAGAGAGCAGTC-3′ and 5′-GGGTGTTTGGTGTCATAGGG-3′.
Results
We previously showed that YB-1 directly binds to the p16 promoter and represses its transcription (26). It was reported that YB-1 belongs to the H1.2 complex which is involved in the repression of p53-mediated transcription (25); therefore, we hypothesized that H1.2 may be involved in the repression of p16. To examine this, we first determined the levels of H1.2 expression in response to the forced expression of an oncogenic form of small GTPase RAS (called oncogenic RAS), which activates the p16 transcription. WI38 cells stably expressing oncogenic RAS (H-RASG12V) were established by retroviral transduction. Immunoblotting showed that p16 protein levels were increased by expressing oncogenic RAS (Figure 1A). In contrast, the levels of H1.2 protein were markedly decreased (Figure 1A). These data indicated that H1.2 expression is down-regulated by expressing oncogenic RAS and that this decrease in H1.2 levels is associated with an increase in p16 expression, suggesting that H1.2 negatively regulates p16 expression. We next examined the involvement of H1.2 in p16 repression. To silence H1.2 expression, we transfected WI38 cells with siRNA oligonucleotides targeting H1.2. Immunoblotting showed that these siRNA oligonucleotides reduced H1.2 levels to an undetectably low level (Figure 1B). Associated with H1.2 reduction was a substantial increase in p16 protein (Figure 1B). qRT-PCR showed that silencing H1.2 increased the p16 mRNA levels by approximately twofold compared with control cells, but not ARF mRNA, which is an alternative reading frame of p16 (Figure 1C). These results supported that H1.2 represses p16 transcription.
Silencing of H1.2 increases the levels of p16 protein and mRNA expression. (A) H-RASG12V was ectopically expressed in WI38 cells by retroviral infection. The expression levels of H1.2, p16, and a-tubulin protein were determined by immunoblotting. (B, C) WI38 cells were transfected with siRNA oligonucleotides targeting H1.2 (H1.2-i) or control siRNA oligonucleotides (Ctr-i). At 72 h after transfection, cells were harvested and subjected to immunoblotting to determine H1.2, p16, and a-tubulin protein levels (B) and q-RT-PCR to determine the expression levels of p16 and ARF mRNA (C). The result of (C) is represented relative to the corresponding values for control cells that were transfected with control siRNA oligonucleotides (Ctr-i). The mean values and standard deviations were calculated from triplicates of representative experiments. The statistical analysis was performed by two-tailed t-test. A p-value of less than 0.05 was considered to indicate a statistically significant difference. *p<0.05. n.s.: Not significant.
Histone H1s including H1.2 are located on chromatin and act as a linker of nucleosomes, leading to transcriptional repression. Therefore, we next examined whether H1.2 associates with the p16 locus. Chromatin immunoprecipitation (ChIP) assays revealed that an H1.2 antibody, but not control IgG, precipitated DNA fragments of the p16 promoter (Figure 2A and B), indicating that H1.2 binds to the p16 promoter. Kim et al. showed that a transcription factor, YB-1, belongs to the H1.2 complex (25). We previously reported that YB-1 binds to and represses the p16 transcription (26). These findings led us to test whether YB-1 is required for the binding of H1.2 on the p16 promoter. ChIP-qPCR demonstrated that silencing YB-1 causes substantial loss of H1.2 binding on the p16 promoter (Figure 2C), suggesting that H1.2 binds to the p16 promoter in a YB-1-dependent manner.
H1.2 binds to the p16 promoter in a YB-1-dependent manner. (A) A schematic representation of the human p16 locus and amplicons (a and b) used for ChIP assays. (B) Binding of H1.2 to the p16 promoter was determined by ChIP assay using control IgG and antibodies against H1.2. PCR was performed by using primers for each amplicon and the products were subjected to agarose gel electrophoresis. (C) The amount of H1.2 binding on the p16 promoter were determined by q-PCR following ChIP assay using antibodies against H1.2 in WI38 cells transfected with control or YB-1 siRNA oligonucleotides. qPCR was performed by using the primer for amplicon b. The result was expressed relative to the corresponding values for control cells that were transfected with control siRNA oligonucleotides (Ctr-i). The mean values and standard deviations were calculated from triplicates of representative experiments. The statistical analysis was performed by two-tailed t-test. A p-value of less than 0.05 was considered to indicate a statistically significant difference. *p<0.05.
To further confirm the repressive role of YB-1 and H1.2 in the p16 transcription, we examined the effect on the expression levels of H1.2 and p16 by silencing YB-1. Immunoblotting showed that silencing YB-1 remarkably increased p16 protein (Figure 3A), as we previously demonstrated (26). Interestingly, silencing YB-1 caused a reduction in H1.2 protein (Figure 3A), but not in H1.2 mRNA (Figure 3C). Collectively, these results suggested that H1.2 expression is post-transcriptionally up-regulated by YB-1, leading to repression of p16 transcription.
Silencing YB-1 results in a decrease in H1.2 expression. (A) WI38 cells were transfected with siRNA oligonucleotides targeting YB-1 (YB-1-i) or control siRNA oligonucleotides (Ctr-i). At 72 h after transfection, cells were harvested and subjected to immunoblotting to determine the effects of YB-1 silencing on H1.2 and p16 expression. (B) H1.2 mRNA levels were determined by q-RT-PCR. The results are expressed as in Figure 1C. The statistical analysis was performed by two-tailed t-test. A p-value of less than 0.05 was considered to indicate a statistically significant difference. n.s.: Not significant.
Discussion
In this study, we showed that H1.2 is involved in the repression of p16 transcription. We showed that H1.2 protein levels were decreased by oncogenic RAS and were inversely correlated with p16 protein levels (Figure 1A). The molecular mechanism underlying the reduction in H1.2 protein by oncogenic RAS remains to be elucidated. We previously reported that oncogenic RAS decreased YB-1 expression, leading to the induction of p16 (26). Silencing YB-1 caused a decrease in H1.2 protein levels, but not mRNA levels (Figure 3A and B). It has been reported that YB-1 functions as an activator of cap-independent translation for SNAIL and other developmentally regulated transcription factors (29). Taken together, these findings suggest a model in which YB-1 reduction by oncogenic RAS leads to decreased H1.2 translation.
The INK4 locus encodes two tumor suppressor genes, p16 and ARF, which positively regulate the pRB and p53 pathways, respectively. We found that silencing H1.2 increased the p16 mRNA, but not ARF mRNA (Figure 1C), suggesting that H1.2 selectively represses the transcription of the INK4 locus. ChIP assay showed that H1.2 associates with the p16 promoter (Figure 2A and B). A biochemical study revealed that YB-1 and p53 are involved in the H1.2 complex (25). These factors bind the promoters of target genes by recognizing specific DNA sequences. H1.2 may be recruited onto the p16 promoter by these factors. Thus, YB-1 may regulate the H1.2 by promoting its translation and binding on the p16 promoter.
p16 plays a pivotal role in tumor suppression by causing cell cycle arrest at the G1 phase. Indeed, inactivation of p16 gene by transcriptional repression and mutation are observed in a wide range of human cancers (14). Furthermore, high expression of the repressors of p16 transcription, such as polycomb proteins, long noncoding RNA ANRIL and YB-1 is observed in several human cancers and is related to a decrease in p16 expression (17, 30-33). Thus, the disruption of p16 repression by H1.2 and YB-1 may lead to tumorigenesis.
Acknowledgements
This work was supported in part by JSPS KAKENHI grant number 22K07160 (to YK), the Naito Foundation (to YK) and Takeda Science Foundation (to YK). We thank H. Nikki March, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Footnotes
Authors’ Contributions
Conceptualization and design, Y.K., Y.T., and R.T.; Supervision, Y.K.; Materials, Y.K.; Data collection, Y.K., Y.T., and R.T.; Analysis, Y.K., Y.T., and R.T.; Writer; Y.K.
Conflicts of Interest
None to be declared.
- Received May 15, 2023.
- Revision received June 6, 2023.
- Accepted June 13, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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