ABSTRACT
Background: 5-Methylcytosine can be oxidized into 5-hydroxymethylcytosine (5hmC) in the genome. Methylated-P16 (P16M) can be oxidized into completely hydroxymethylated-P16 (P16H) in human cancer and precancer cells. The aim of this study is to investigate the biological function of P16H.
Methods: True P16M and P16H were analyzed using bisulfite/TAB-based assays. A ZFP-based P16-specific dioxygenase (P16-TET) was constructed and used to induce P16H. Cell proliferation and migration were determined with a series of biological analyses.
Results: (A) The 5hmCs were enriched in the antisense-strand of the P16 exon-1 in HCT116 and AGS cells containing methylated-P16 alleles (P16M). (B) P16-TET induced both P16H and P16 demethylation in H1299 and AGS cells and reactivated P16 expression. Notably, P16H was only detectable in the sorted P16-TET H1299 and AGS cells that did not show P16 expression. (C) P16-TET significantly inhibited the xenograft growth derived from H1299 cells in NOD-SCID mice, but did not inhibit the growth of P16-deleted A549 control cells. P16-siRNA knockdown could rescue P16-TET-inhibited cell migration.
Conclusion: Hydroxymethylated P16 alleles are transcriptionally inactive.
Significance: This study demonstrates for the first time that the hydroxymethylated P16 alleles are transcription-inactive.
INTRODUCTION
It is well known that ten-eleven translocation methylcytosine dioxygenases (TET-1/2/3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in the genome [1-4]. While oxidation of 5mC leads to active DNA demethylation, a certain proportion of 5hmC sites remain in the genome with a strand-asymmetric and strand-symmetric distribution pattern that provides its own regulatory function [5-9]. Although it is frequently reported that the 5hmC level of some genes is positively correlated with increased gene expression, it is not clear whether 5hmC itself or related DNA demethylation contribute to the reactivation of gene transcription.
Typical bisulfite-based assays cannot discriminate 5mCs from 5hmCs. The classic term “DNA methylation” is, in fact, total DNA methylation, including true methylation and hydroxymethylation. Total methylation of the CpG island (CGI) flanking the transcription start site (TSS) in the P16 gene (CDKN2A) is prevalent in human cancer and precancerous tissues [10,11] and is linked to increased cancer development from epithelial dysplasia in many organs [12-18]. P16 methylation (P16M) not only directly inactivates P16 transcription [19] but also represses ANRIL transcription [20]. Our recent study demonstrated that there were dense 5hmCs in the P16 exon-1 CGI in HCT116 cells, and no mRNA transcripts from the hydroxymethylated P16 (P16H) alleles were detected in the cells [21,22]. P16H was detected in 9.3% of human oral epithelial dysplasia (OED) tissues [23]. However, the malignant transformation risk was similar between P16M-positive OED patients with and without P16H. It is a fundamental question in epigenetic research to clarify whether hydroxymethylation of TSS-flanking CGIs leads to transcriptional activation of genes.
In this study, we characterized the distribution patterns of 5hmCs within the sense and antisense strands (S- and AS-strands) of the P16 promoter and exon-1 CGIs using detailed TET-assisted bisulfite (TAB)-based assays, and found that 5hmCs were enriched in the AS-strands of P16 exon-1 CGIs in cancer cells. To elucidate the possible role of P16H, a P16-specific TET-1 was constructed and used to induce P16H in cancer cell lines. Notably, our data showed, for the first time, that P16H itself could not reactivate gene transcription.
METHODS
Cell Lines and Culture
The colon cancer cell line HCT116 was purchased from the American Type Culture Collection (ATCC). The GC cell line AGS and the lung cancer cell line H1299 were kindly provided by Prof. Chengchao Shou from the Peking University Cancer Hospital and Institute. The colon cancer cell line, RKO was kindly provided by Prof. Guoren Deng from the University of California, San Francisco. These cells were cultured in RPMI 1640 containing 10% FBS and 100 U/mL penicillin/streptomycin (Invitrogen, California, USA) at 37°C in a humidified incubator with 5% CO2.
These cell lines were tested and authenticated by Beijing JianLian Genes Technology Co., LTD before they were used in this study. STR patterns were analyzed using a Goldeneye™20A STR Identifiler PCR Amplification Kit. Gene Mapper v3.2 software (ABI) was used to match the STR pattern with the ATCC online databases.
Characterization of 5mC and 5hmC Sites in P16 CGIs
Total P16M was analyzed using 150-bp regular methylation-specific PCR (MSP) [24]. To selectively detect P16H, the genomic DNA (3 μg), spiked with M.sssI-methylated and 5hmC-containing λ-DNA controls, was modified using the TET-Assisted Bisulfite (TAB) Kit, according to manufacturer’s instructions (WiseGene, Cat# K001). During TAB-modification, 5mC was oxidized to 5caC, and both 5caC and unmethylated cytosine were subsequently converted to uracil through bisulfite-induced deamination, whereas 5hmC was protected from oxidation via 5hmC-specific β-glucosylation [25]. The conversion rates of unmethylated cytosine, 5mCs, and 5hmCs in the bisulfite-/TAB-treated λ-DNA controls were 100%, 99.7%, and 1.5%, respectively (Figure S1). P16H was analyzed using the TAB-modified templates with MSP (TAB-MSP).
The proportion of hydroxymethylated S- and AS-strands of the P16 promoter and exon-1 CGIs were analyzed using DHPLC and clone sequencing, respectively [26,27]. The adjusted ratio of the peak height for the hydroxymethylated region to that of the unmethylated region was used to represent the P16H proportion that was adjusted. The ratio of the P16M peak height to the P16U peak height for P16-hemimethylated HCT116 cells was used as a reference. The sequences of the universal primers used to amplify these fragments are listed in Table S1.
Construction of Expression Vectors and Transfection
To construct the P16-specific DNA dioxygenase (P16-TET) expression vector, an SP1-like engineered seven-zinc finger protein (7ZFP-6I) [19] that can specifically bind to the 21-bp fragment (5’-gaggaaggaaacggggcgggg-3’, including an Sp1-binding site) within the human P16 core promoter [28,29], was fused with the catalytic domain (CD: 1418-2136 aa) of human TET1 (NM_030625.2) [30] and inserted into a pcDNA3.1b vector and then used in transient transfection assays. An inactive P16-TET mutant containing an H1671Y mutation in the CD domain vector was also constructed and used as a negative control vector (Figure S2A). The P16-TET sequence was further integrated into the expression-controllable pTRIPZ vector carrying a “Tet-on” switch (Open Biosystem, USA) (Figure S2B) [19]. Purified P16-TET pTRIPZ plasmid was mixed with VSVG and Δ8.9 (Addgene, USA) to prepare lentivirus transfection particles. The fresh lentivirus particles were used to stably infect AGS and H1299 cells containing homogenously methylated P16 CpG islands. Doxycycline (Dox; final conc. 0.25 μg/mL) was added to the medium to induce P16-TET expression.
Two P16-specific siRNAs (5’-ccgua aaugu ccauu uauatt-3’ and 5’–uauaa augga cauuu acggtt-3’) were synthesized (GenePharma, Shanghai) and used to transiently transfect cells at a final concentration of 1.0 μg/mL. Two scrambled siRNAs (5’-uucuc cgaac guguc acgutt-3’ and 5’-acgug acacg uucgg agaatt-3’) were used as negative controls (NC).
Treatment of 5’-Aza-Deoxycytidine (DAC)
The AGS cells were treated with DAC (final concentration 20 nM; Abcam ab120842, Cambridge, UK) for 7 days in the P16-immunostaining assay or 10 days prior to FACS sorting.
Extraction of RNA and Quantitative RT-PCR (qRT-PCR)
Cells were harvested when they reached a confluency of approximately 70%. Total RNA was extracted by TRIzol (Invitrogen, California, USA). The cDNA was reverse-transcribed using the ImProm-II™ Reverse Transcription System (A3800; Promega). The expression levels of the ANRIL, P16, P15, P14, and TET-1/2/3 genes were analyzed by quantitative RT-PCR using the corresponding primer sets (Table S1), as previously described [20]. Power SYBR Green PCR Master Mix (Fermentas, Canada) was used in the qRT-PCR analyses (ABI-7500FAST). The relative mRNA level was calculated based on the average Ct value of the target gene and the Alu reference [2-(Cttarget_gene-CtAlu)] [31].
Western Blot and Confocal Microscopy Analysis of the P16 Expression Status
The P16 mRNA and protein levels in the cells were analyzed as previously described [19]. Rabbit monoclonal antibody against human P16 protein (ab108349, Abcam, Britain) was used in the Western blot assay, and mouse monoclonal antibody against the human P16 protein (Ventana Roche-E6H4, USA) was used in the immunostaining assay.
Cell FACS Sorting
The P16-TET stably transfected H1299 cells (treated with doxycycline for 21 days) and AGS cells (treated with 5-aza-deoxycytidine for 10 days) were fixed with methanol, permeabilized with 0.1% Tween-20 in PBS, pretreated with 10% fetal bovine serum and 0.3 M glycine in PBS, and were then stained with the mouse monoclonal antibody against the human P16 protein (Ventana Roche-E6H4, USA) and the FITC-tagged secondary antibody. The P16-staining cell population proportion was determined using an immuno-fluorescence confocal microscope. These cells were sorted by FACS and divided into three subpopulations, strong-, weak-, and non-P16-staining, using P16-TET H1299 cells without doxycycline treatment or AGS cells without DAC treatment as P16 protein negative controls. According to the confocal analysis results, we setup the cutoff value to sort definite and indefinite P16 protein positive (P16(+) and P16(±)) cell subpopulations. The strong and weak FITC-staining cells were called as the P16(+) and P16(±) subpopulations, respectively.
IncuCyte ZOOM and Transwell Migration Tests
The long-term live content kinetic imaging platform (IncuCyte Zoom, Essen BioSci, USA) was used to dynamically detect the proliferation and migration of live cancer cells. The phase object confluence (%) was used to generate a cell proliferation curve. The relative wound density, a measure (%) of the density of the wound region relative to the density of the cell region, was used as the metric for cell migration. The transwell migration test was performed as previously described [19].
Xenografts in SCID Mice
Cells stably transfected with the P16-TET vector were induced with 0.25 μg/mL doxycycline for 7 days and then subcutaneously injected into one lower limb of each NOD-SCID mouse (105 cells/injection; female, 5 weeks old, 10∼20 g, purchased from Beijing Huafukang Biotech). The negative control cells stably transfected with the empty pTRIPZ vector were simultaneously injected into the opposite side of each mouse. These mice were given distilled, sterile water containing 2 μg/mL doxycycline and were sacrificed on the 50th post-transplantation day. The xenografts were weighed and histologically confirmed [19]. Two repeat experiments were performed.
Statistical Analysis
Student’s t-test was used for statistical analysis. All P-values were two-sided, and a P-value of <0.05 was considered to be statistically significant.
RESULTS
Characterization of 5hmCs in the P16 Exon-1 CGI
We recently found that there were dense 5hmCs in the P16 exon-1 in HCT116 cells [21], in which the wildtype P16 alleles are silenced by DNA methylation and the mutant alleles containing a G-insertion in exon-1 are unmethylated. To characterize the distribution pattern of 5hmCs in the P16 CGI, the S- and AS-strands of the P16 promoter and the exon-1 regions were amplified using conventional bisulfite-modified and TAB-modified single-strand DNA samples from HCT116 cells as templates. Next, the proportions of total P16M- and P16H-containing fragments were quantitatively analyzed by DHPLC. As expected, the total P16M peak and the P16U peak were both detected in the all bisulfite PCR products from both the S- and AS-strands of the P16 promoter and exon-1 fragments (Figure 1A-D: HCT116, left charts). However, a high P16H peak was detected only in the exon-1 AS-strand and the P16H proportion reached up to 88% (=0.77/0.87) (Figure 1D: HCT116_TAB, left chart). In the promoter AS-strand fragment, the P16H peak was very low (Figure 1C: HCT116_TAB, left chart). In the S-strands of the promoter and exon-1 fragments, much lower levels of the P16H peaks were detected (Figures 1A and 1B: HCT116_TAB, left charts).
The TAB sequencing results for the HCT116_TAB PCR products confirmed the DHPLC analysis results (Figure 1A-D: right charts). Dense 5hmCs were found in the wildtype exon-1 AS-strand (tracked with a G-deletion; 5hmC-density, 82.9%), but not in the paired S-strand (5hmC-density, 17.6%). No clone containing more than one 5hmC was detected in the promoter AS-strand (5hmC density, 2.0%). Sporadic 5hmCs were distributed in the promoter S-strand (5hmC density, 22.3%). Together, the results of the TAB-DHPLC and TAB sequencing analyses consistently demonstrated that 5hmCs were enriched mainly in the AS-strand of the wild-type P16 exon-1 in HCT116 cells. This indicates that wild-type P16 exon-1 is hydroxymethylated mainly in the AS-strand and is methylated truly in the S-strand in HCT116 cells. As described below, dense 5hmC sites were also detected in the AS-strand of the P16 exon-1 in gastric cancer AGS cells.
Construction of Engineered P16-TET
To study whether P16H affects gene transcription, an expression controllable P16-specific dioxygenase pcDNA3.1-vector (P16-TET) and its inactive mutant control vector were constructed through fusing an engineered P16 promoter-specific seven zinc finger protein (7ZFP-6I) [28] with the catalytic domain of human TET1 (Figure S2A). H1299 cells were chosen because epigenetic editing of the methylated P16 CGIs by the P16-specific transcription factor (P16-ATF; 7ZFP-6I-VP64) has been optimized in this cell type [28]. As expected, the results of both qRT-PCR and immunofluorescence staining showed that the methylated P16 alleles were re-activated in H1299 cells 6 days after transient transfection with the P16-TET pcDNA3.1-vector (Figure 2). Such P16 reactivation was not observed in the P16-TET mutant control cells. This indicates that P16-TET is P16 gene reactive and could be used in further studies.
Induction of P16H by P16-TET
To study the possible biological functions of P16-specific hydroxymethylation, the P16-TET coding sequence was further integrated into the pTRIPZ lentivirus vector carrying a “Tet-on” switch to allow the gene expression to be controlled for stable transfection (Figure S2B). In the P16-TET stably transfected H1299 cells, the results of the TAB-MSP analysis showed that P16H signals appeared in the P16-TET cells 3 days after Dox induction (P16-TET&Dox_3d; Figure 2A, TAB-MSP), but did not appear in cells transfected with the empty vector (control cells with Dox treatment) (Vector&Dox_14d) or in baseline P16-TET cells without Dox induction, in which only nonhydroxymethylated P16 alleles (P16N) were detected. In the MSP analysis, P16U was detectable in the P16-TET&Dox cells 3 days following Dox induction (Figure 3A, MSP). The bisulfite-DHPLC results showed that a low P16U peak was detected beginning on the 14th day (Figure S3A, red-arrow). Two P16U clones were also observed on the 28th day from the bisulfite sequencing (Figure S3B, red-star). These results indicate that both P16H and P16U were induced in the P16-TET&Dox cells.
Furthermore, the Western blot results revealed that P16 protein was detected in the P16-TET&Dox cells since the 7th day, but not in the Vector&Dox control cells (7d; Figure 3B). The qRT-PCR results showed a weak reactivation of P16 transcription beginning on the 4th day (Figure 3C). The immunofluorescence confocal microscopy results confirmed the presence of P16 protein in the nuclei of H1299 cells (Figure 3D). In addition, the expression status of the control genes P15 and P14 was not affected, whereas the expression level of ANRIL, which is coordinately expressed with P16, was increased (Figure S4). This suggests a high specificity for the zinc finger protein-based P16-TET to induce P16H and P16U.
Similarly, on the 7th day after Dox induction, transcriptional reactivation of P16 was also observed in P16-TET stably transfected gastric cancer AGS cells, in which P16 alleles are homogenously methylated (Figures 4A-4E). Interestingly, P16U signals were not detected in P16-TET AGS cells after Dox induction for 11 days (P16-TET&Dox_11d) in the bisulfite-DHPLC and bisulfite sequencing analyses (Figure 4A and 4C). P16H signals were observed in the TAB-DHPLC and TAB sequencing results (Figure 4B and 4D), indicating that hydroxymethylation occurred earlier than demethylation at P16 CGIs. A few baseline 5hmCs were also found in the P16 exon-1 AS-strand of AGS mock control cells. Although weak P16 mRNA signals were detected in P16-TET AGS cells after Dox induction for 7 days and 11 days according to sensitive RT-PCR analysis (Figure 4E), P16 protein was not detected in these cells according to the insensitive Western blot analysis (Figure 4F).
Transcription Silencing of P16 alleles by Hydroxymethylation
To clarify whether DNA hydroxymethylation or demethylation contributes to P16 reactivation, we further analyzed the hydroxymethylation status of P16 CGIs in cell subpopulations with strong, weak, and no P16 staining (P16(+), P16(±), and P16(-)) that were sorted from P16-TET&Dox_21d H1299 cells (Figure 5A). Interestingly, P16H signal was detected only in the P16(-) subpopulation, but not in the P16(+) and P16(±) subpopulations in the TAB-MSP analysis (Figure 5B). TAB sequencing also showed dense 5hmCs among 3 of the 14 clones (21.4%) of the exon-1 AS-strand TAB-PCR products from the P16(-) subpopulation, with an average hydroxymethylation density of 95.2% for these 3 clones (Figure 5C). The occurrence of 5hmCs in the promoter AS-strand was not detected in the TAB-DHPLC and TAB sequencing results (data not shown).
The above results were further confirmed in AGS cells. As described above, P16 protein could not be detected in P16-TET&Dox AGS cells after Dox treatment for 11 days (Figure 4F). To obtain a P16(+) AGS subpopulation by FACS, the DNA methyltransferase inhibitor 5-aza-deoxycytidine (DAC, final concentration 20 nM) was used to increase the P16 protein level within P16-TET AGS cells. In the immunostaining cell analysis, nucleic P16 protein was detected in 3.5% of P16-TET AGS cells after DAC treatment for 10 days (P16-TET&DAC_10d, with baseline P16-TET expression without Dox induction), while nucleic P16 protein was detected in only 0.5% of the AGS cells treated with DAC alone (Figure S5). Next, the P16(+), P16(±), and P16(-) subpopulations were sorted from these P16-TET&DAC_10d AGS cells (Figure 6A). Once again, the P16H signal was detected only in the P16(-) subpopulation, and not in the P16(+) and P16(±) cells by the TAB-MSP and TAB-DHPLC assays (Figures 6B and 6C). In contrast, P16N signal was detected in all three subpopulations. The TAB sequencing results confirmed this. Dense 5hmCs were observed in the P16 exon-1 AS-strand in the P16(-) subpopulation, but not in the P16(+) subpopulation (Figure 6E).
Collectively, the above results indicate that P16H occurs only in P16(-) cells, and not in P16(+) and P16(±) cells, suggesting that the P16H alleles should be transcriptionally inactive.
P16 Allele-Dependent Inhibition of Tumor Growth by P16-TET
Although a proliferation difference was not observed between the P16-TET and control vector, which were stably transfected in H1299 cells in vitro (Figure 7A), the average weight of tumor xenografts (n=8) of the P16-TET stably transfected cells was significantly lower than that of the control cells in NOD-SCID mice on the 50th post-transplantation day (P<0.001, Figures 7B and 7C). Morphologic differences were not observed between P16-TET and control vector xenografts (Figure 7D). This result was confirmed in a repeat experiment (Figure S6A).
Meanwhile, this difference could not be observed in xenograft tumors from lung cancer A549 control cells in which the P16-P15-P14 alleles were homogeneously deleted (Figure S6B). These data suggest that P16-TET may specifically inhibit the growth of cancer cells in vivo in a P16 allele-dependent manner.
Although P16-TET did not affect the proliferation of H1299 cells in vitro, the results of the IncuCyte ZOOM wound-scratch and typical transwell assays showed that P16-TET significantly inhibited H1299 cell migration (Figures S7A and S7B). In a rescue assay, P16 siRNA-knockdown significantly reversed the inhibited migration of the P16-TET&Dox H1299 cells (Figure S7C). These results provide further evidence to support that P16-TET may inhibit cell migration through P16 reactivation.
DISCUSSION
DNA hydroxymethylomes at the base-resolution level have been analyzed in embryonic stem cells, adult tissues, and tumors [32-38]. Many functions of DNA hydroxymethylation in the genome have been illustrated by TET-1/2/3 knockout studies [5-7,34,38,39]. However, the actual effect of hydroxymethylation of CGIs on gene transcription remains elusive. In the present study, we demonstrated that 5hmCs were enriched in the AS-strand of the P16 exon-1 CGI. Most importantly, this study showed for the first time that DNA hydroxymethylation itself could not reactivate P16 gene transcription. Instead, hydroxymethylation-mediated active DNA demethylation could reactivate P16 gene transcription, which subsequently inhibited the migration and growth of cancer cells in vivo.
It is well known that an appropriate proportion of 5hmCs in the genome is distributed with a strand bias [9,35]. We recently reported that there were dense 5hmCs in the P16 exon-1 AS-strands in HCT116 cells [21,22]. Based on the comprehensive TAB-DHPLC and TAB sequencing results, here, we further demonstrated that 5hmCs were enriched only in the AS-strand of the P16 exon-1 in HCT116 cells and AGS cells, while sporadic 5hmCs were detected in the S-strand of P16 promoter and exon-1 regions. The fact that 88% of the exon-1 AS-strand CpGs in the wild-type P16 alleles in HCT116 cells are hydroxymethylated indicates that P16 exon-1 has a methylation: hydroxymethylation (M:H) mixture, composed of a fully hydroxymethylated AS-strand and a truly methylated S-strand.
It has been reported that triple knockout of TET-1/2/3 led to bivalent promoter hypermethylation in H1 cells [40]. Through re-analyzing four publicly available hydroxymethylome datasets for H1 cells, brain tissue, kidney tumor and paired normal tissues [9,35,36], we found that most 5hmCs in the 5’-untranslated regions (5’UTRs) were enriched at CGI CpGs in the kidney tumor and normal tissues with low global 5hmC/5mC ratios (2.7-9.4%), but were enriched in non-CGI CpGs in H1 cells and the brain tissue with high 5hmC/5mC ratios (26.2-57.0%). Similar phenomena were also observed in the promoter regions. However, most 5hmCs were located at non-CGI CpGs in the gene body, 3’UTR, and downstream regions in all four samples (Han X, et al., prepared for publication). These observations imply that 5’UTR CGI CpGs may be intrinsically prone to hydroxymethylation or resistant to hypermethylation. This may account for the 5hmC enrichment in P16 exon-1 AS-strands.
TSSs are DNA replication start sites. S- and AS-strands of genes are generally replicated by different types of DNA polymerases in eukaryotic cells (Polδ for the leading strand and Polα for the lagging strand). Unlike true DNA methylation that is maintained by DNMT1 during DNA synthesis in the S-phase of the cell cycle, DNA hydroxymethylations are probably maintained by the de novo methyltransferases DNMT3a/b [41]. It is of great interest to study the mechanisms leading to the strand bias of DNA hydroxymethylation.
Three types of epigenetic editing methods, including ZFP-, transcription activator-like effector (TALE)-, and CRISPR/dCas9-based systems, have emerged as advanced tools to study the functions of epigenetic modifications [19,42-46]. According to the reported data, the specificity and efficiency of ZFP-based epigenetic editing tools are likely higher than those of TALE-based editing tools or CRISPR/dCas9-based editing tools. For example, the expression controllable ZFP-based P16-Dnmt could selectively methylate entire P16 CGIs around the TSS [19]. However, CRISPR/dCas9-Dnmt3a, combined with P16-sgRNA, could specifically methylate only approximately 50 bp sgRNA target-flanking sequences (not including the sgRNA target) [47,48]. In contrast, P16 TALE-Dnmt could methylate the P16 target and other CGIs within the P15-P14-P16MTAP gene cluster and repress their transcription, with low specificity [49]. We recently reported that ANRIL expression was repressed in cancer cells by P16 methylation [20]. Here, we further demonstrated that the ANRIL expression was upregulated in the P16-TET-expressing cells and that the mRNA levels of P15 and P14 were not increased. These observations suggest that P16-TET could specifically demethylate P16 CGIs via DNA hydroxymethylation and reactivate the transcription of both the P16 and ANRIL genes.
Recently, we found that all P16 mRNA clones in the HCT116 cells were transcribed only from the unmethylated P16 alleles, and none from the methylated: hydroxymethylated (M:H) P16 alleles [21], and that both true P16M and P16H could similarly increase the risk for malignant transformation of oral epithelial dysplasia in a prospective study [23]. The findings of the present study show that the P16-TET-induced hydroxymethylation of P16 alleles in both H1299 and AGS cells retain transcriptional silence, which provides a possible mechanism to explain the above observations.
There are many differences between cell culture and animal models. Although the proliferation of H1299 cells that are stably transfected with P16-TET was not changed under in vitro culture conditions, the growth of xenograft tumors from these cells was obviously inhibited in host mice. The exact reasons leading to this difference are unknown; however, the reactivation of methylated P16 alleles via DNA demethylation by P16-TET may account for the growth inhibition in vivo. The growth inhibition of xenograft tumors from the P16-deleted A549 control cells was not observed, suggesting that the growth inhibition of xenografts by P16-TET may be a P16-dependent phenomenon. In the rescue assay, siRNA knockdown of P16-TET-reactivated P16 expression almost completely reversed the inhibition of P16-TET-induced cell migration. This further suggests that the inhibition of the cancer cell migration by P16-TET may be a P16-specific effect.
In conclusion, we found that hydroxymethylation of P16 CGI is located mainly in the exon-1 AS-strand. P16H alleles are transcriptionally inactive. P16 demethylation via hydroxymethylation could reactivate gene transcription and inhibit the growth of cancer cells.
Disclosure of potential conflicts of interest
The authors have declared that no competing interests exist.
Funding
Funding for this project was from the National Natural Science Foundation of China (81672770), Beijing Municipal Commission of Health and Family Planning (PXM2018_026279_000005), Beijing Municipal Administration of Hospital Clinical Medical Development of Special Funding Support (XM201303), and Beijing Science and Technology Commission (Z151100001615022) to DD.
Author contributions
YG and PL: elucidated the biological function of P16 hydroxymethylation; SQ: discovered P16 hydroxymethylation and its association with gastric carcinogenesis; XH: demonstrated the strand-bias distribution of 5hmCs in the P16 alleles; CC, Z-mL, and BZ: constructed the P16-specific oxygenase; LG performed the animal experiments; BZ performed immunostaining and cell sorting assays and codesigned the study; ZL and JZ: carried out other experiments; DD: designed the study, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We thank Mr. Jordan M. Grainger (Predoctoral student, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA) and Dr. Huidong Shi (Cancer Center, Georgia Medical College, Augusta, USA) for English language editing.
Footnotes
↵# Equal contribution
Author contributions YG and PL: elucidated the biological function of P16 hydroxymethylation; SQ: discovered P16 hydroxymethylation and its association with gastric carcinogenesis; XH: demonstrated the strand-bias distribution of 5hmCs in the P16 alleles; CC, Z-mL, and BZ: constructed the P16-specific oxygenase; LG performed the animal experiments; BZ performed immunostaining and cell sorting assays and codesigned the study; ZL and JZ: carried out other experiments; DD: designed the study, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.