Independent functions of DNMT1 and USP7 at replication foci
© The Author(s) 2018
Received: 21 December 2017
Accepted: 16 February 2018
Published: 27 February 2018
It has been reported that USP7 (ubiquitin-specific protease 7) prevents ubiquitylation and degradation of DNA methyltransferase 1 (DNMT1) by direct binding of USP7 to the glycine-lysine (GK) repeats that join the N-terminal regulatory domain of DNMT1 to the C-terminal methyltransferase domain. The USP7-DNMT1 interaction was reported to be mediated by acetylation of lysine residues within the (GK) repeats.
We found that DNMT1 is present at normal levels in mouse and human cells that contain undetectable levels of USP7. Substitution of the (GK) repeats by (GQ) repeats prevents lysine acetylation but does not affect the stability of DNMT1 or the ability of the mutant protein to restore genomic methylation levels when expressed in Dnmt1-null ES cells. Furthermore, both USP7 and PCNA are recruited to sites of DNA replication independently of the presence of DNMT1, and there is no evidence that DNMT1 is degraded in cycling cells after S phase.
Multiple lines of evidence indicate that homeostasis of DNMT1 in somatic cells is controlled primarily at the level of transcription and that interaction of USP7 with the (GK) repeats of DNMT1 is unlikely to play a major role in the stabilization of DNMT1 protein.
The structures of several mouse and human DNMT1 proteins have been determined [2, 4–6], and in all cases, the (GK) repeats are disordered in the crystal structure and not resolved, which implies that they do not form stable associations with other domains of DNMT1. The approximate positions of the (GK) repeats with respect to other domains of DNMT1 are shown in Fig. 1d. It has been reported that the (GK) repeats are involved in controlling proteasomal degradation of DNMT1 via reversible acetylation of lysines within the (GK) repeats [7–11]; this has been suggested to couple DNMT1 biosynthesis and degradation to the cell cycle . Ubiquitin-specific protease 7 (USP7; also known as Herpes-associated ubiquitin-specific protease or HAUSP) [12–14] has been reported to bind to the unacetylated (GK) repeats of DNMT1 ; the acetylated form was reported to be incapable of binding to USP7 in vitro and has been proposed to undergo ubiquitylation at lysine 586 (RFTS domain) and lysine 997 (BAH2 domain) , and to be targeted for proteasomal degradation . DNMT1 was also reported to be required for the recruitment of USP7 to sites of DNA replication in S phase nuclei . These reports depended on the results of in vitro studies or transfection of tagged and mutated forms of DNMT1 into cells that contained endogenous DNMT1; the relative levels of endogenous and exogenous DNMT1 were not reported and only exogenous DNMT1 was observed. Furthermore, levels of DNMT1 do not change appreciably during the cell cycle , as shown in Fig. 1a, although DNMT1 is not expressed in G0 cells . We report here that reduction in USP7 to undetectable levels in mouse and human cells did not cause a measurable reduction in content of DNMT1 or in DNA methylation, that substitution of the acetylated lysines within the (GK) repeats by glutamines does not affect the amount or activity of endogenous DNMT1, and that recruitment of USP7 to replication foci during S phase is independent of the presence of DNMT1. These and several other lines of evidence indicate that any USP7-DNMT1 interaction does not play a major role in the stabilization of DNMT1 and that levels of DNMT1 are regulated at the level of transcription.
Mouse ES cells as described in  were cultured on gelatin following standard techniques. Stable ES cell lines were generated by nucleofection of Dnmt1-null ES cells  with MT80 minigene and pGKPuro plasmid for Puromycin resistance . MT80 minigene, carrying 12 kb of 5′ Dnmt1 genomic sequence with endogenous promoter and 5.5 kb of Dnmt1 cDNA [19, 20], was modified by the addition of an N-terminal Flag-HA tag after the translation start site. Point mutations were produced using QuikChange Site-Directed Mutagenesis kit (Agilent). Individual clones were selected with Puromycin for 10–14 days and picked into 96-well plates. Clones were genotyped using primers specific to the Flag-HA tag. Positive clones were propagated, and levels of DNMT1 expression were tested by Western blotting. Clones expressing DNMT1 at wild-type levels were used for further studies.
Usp7 cl/+ mice  were intercrossed to generate homozygous conditional mutant embryos. MEFs were derived and transfected with a construct that expressed SV40 large T antigen. MEFs were subsequently infected with Ad–GFP and Ad–Cre–GFP viruses from Vector Biolabs (Catalogue numbers 1761 and 1710). Cultures that showed near-complete infection with Ad-Cre-GFP virus as assessed by visualization of GFP expression were analyzed after five days.
To generate the inducible Usp7 shRNA knockdown cells, H1299 cells were transfected with pTRIPZ encoding a Tet-inducible Usp7 short hairpin RNA obtained from Thermo Open Biosystems (clone ID: V2THS_172409) and a Puromycin resistance cassette. 48 h later Puromycin (5 μg/ml) was added to the transfected cells for 14 days. To induce shRNA transcription, 5 μg/ml of doxycycline was added to the culture medium for 72 h prior to analysis.
Whole cell extracts were prepared by lysis in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Deoxycholic acid, 0.1% SDS, 50 mM Tris pH 7.5) and briefly sonicated to disrupt genomic DNA, then heated to 100° in SDS and loaded onto SDS-PAGE gels. Proteins were transferred to nitrocellulose membrane and blocked in 5% milk, 0.1% Tween 20, PBS for 1 h at room temperature. Blots were incubated at 4 °C overnight with primary antibodies in 10% FBS, 0.1% Tween 20. After incubation with DNMT1 antibody, blots were washed with PBST and PBS. Antibodies used: rabbit polyclonal to DNMT1 (Cell Signaling Technology, 5032 (D63A6) 1:2500 dilution), rabbit polyclonal to USP7 (Bethyl Laboratories, IHC00018; 1:5000 dilution), rabbit polyclonal to HA tag (Abcam, ab9110, 1:5000), rabbit polyclonal to UHRF1 (Bethyl Laboratories, A301-470A, 1:1000), mouse monoclonal to alpha-tubulin (Abcam, ab7291, 1:10,000).
Three independent biological replicates were performed. DNMT1 and tubulin levels were quantified using ImageJ. Statistical analysis was performed using the two-tailed t test.
Genomic DNA was extracted by digestion with proteinase K and RNase A followed by phenol/chloroform extraction and isopropanol precipitation. Genomic DNA was digested for two rounds with methylation-sensitive enzyme HpaII, its isoschizomer MspI as a control, or McrBC (all from NEB). DNA was quantified and ran on 0.8% agarose gel, which was stained with ethidium bromide.
Southern blot analysis was performed with IAP probes generated by PCR. Primers used for probe amplification: probe IAP F (GGTAAACAAATAATCTGCGC); probe IAP R (CTGGTAATGGGCTGCTTCTTCC). DNA in agarose gels was transferred to a Nytran SPC membrane (GE Healthcare) overnight in 10 × SSPE buffer. After crosslinking, membrane was prehybridized with 6X SSC, 5X Denhardts, 1% SDS, 10% Dextran Sulfate for 1 h at 45° and incubated overnight with IAP probe at 45°. Membranes were washed once with 2X SSC, 0.5% SDS; 2X with 1X SSC, 0.5% SDS, and 1X with 0.2X SSC, 0.5% SDS.
Statistical analysis was performed on biological replicates using the two-tailed t test.
ES cells were cultured on glass slides. For the PCNA immunostaining, cells were treated with 0.5% Triton X-100 in CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES [piperazine-N,N-bis(2-ethanesulfonic acid)], pH 6.8, 3 mM MgCl2, 1 mM EGTA) for 30 s at 4 °C, and then treated with methanol for 20 min at − 20 °C .
Cells were permeabilized/blocked in Block solution (5% Donkey serum, 0.3% Triton, 1X PBS) and then incubated overnight with primary antibody diluted in Block solution at 4 °C. The following primary antibodies were used: mouse monoclonal to PCNA (Abcam, ab29, 1:500 dilution), rabbit polyclonal to USP7 (Bethyl Laboratories, IHC00018; 1:100 dilution).
Cells were washed ten times with PBS and incubated for 1 h at room temperature with the following secondary antibodies diluted in Block buffer: Cy3-conjugated donkey anti-mouse (Jackson ImmunoResearch; 1:200 dilution) and IgM Alexa 488–conjugated IgG donkey anti-rabbit (Jackson ImmunoResearch; 1:500 dilution). Slides were subsequently washed with PBS, counterstained with Hoechst 33,342 (Invitrogen) and mounted with Vectashield mounting medium (Vector Labs).
Statistical data were calculated for groups with normal distributions and similar variances. Variation within each group of data is reported as standard deviation. ‘n’ represents the number of biological replicates. p values were calculated using the two-tailed t test. The R program was used to ensure that n = 2 was sufficient to establish a power of 0.8.
Ablation of USP7 does not affect stability or function of DNMT1
Mutation of GK repeats results in normal stability and activity of DNMT1
USP7 localizes to replication foci independently of DNMT1
Several lines of evidence indicate that USP7 does not control cell cycle-dependent levels of DNMT1 in vivo as had been claimed. First, DNMT1 is not degraded after S phase in cycling cells; in Xenopus extracts DNMT1 levels and chromatin loading are unperturbed upon the inhibition of proteasome activity . Second, DNMT1 levels are independent of the presence of USP7, as shown in Fig. 2. Third, substitution of the GK repeats with GQ repeats, which prevents the acetylation of DNMT1, does not affect steady-state levels of DNMT1 (Fig. 3). Fourth, USP7 localizes to replication foci independently of DNMT1 (Fig. 4). Fifth, modification of the endogenous Dnmt1 locus so as to delete the first 118 amino acids of DNMT1 caused the accumulation and persistence of truncated but fully active DNMT1 to ~ 5 times normal levels . This is the only genetically defined region of DNMT1 that affects protein stability. However, this shortened and stabilized form of DNMT1 is produced only in growing oocytes and has not been detected in somatic cells .
The (GK) repeats are both highly basic and unstructured; they are therefore capable of adopting many conformations and would be expected to bind nonspecifically in vitro to many proteins that contain acidic pockets, as is the case for USP7. Furthermore, prior studies expressed tagged recombinant DNMT1 in wild-type cells where the relative amounts of tagged recombinant DNMT1 to endogenous wild-type DNMT1 were not reported .
Despite the numerous reports of post-translational regulation of DNMT1 expression in an acetylation-dependent manner [7–11], strong evidence indicates that DNMT1 levels are normally controlled at the transcriptional rather than the post-translational level. Cells heterozygous for loss-of-function mutations at Dnmt1 express one-half the amount of DNMT1 protein when compared to wild-type cells , and mice that contain additional copies of the Dnmt1 gene overexpress DNMT1 protein . DNMT1 is overexpressed in Friend Murine Erythroleukemia cells as a result of spontaneous amplification of the Dnmt1 gene in this cell type . DNMT1 is present in nuclei at constant levels throughout the cell cycle and is recruited to replication foci during S phase . DNMT1 is down-regulated in G0 cells, but this is likely to occur at the transcriptional level . Treatment of cells with drugs that induce entry into G0 phase will cause a reduction in DNMT1 levels due to cell cycle effects rather than a direct effect on DNMT1 stability . There is no direct evidence of a post-translational mechanism that compensates for reduced or increased DNMT1 transcript levels, and changes in Dnmt1 gene dosage result in proportionate increases or decreases in DNMT1 protein level.
Many studies have tried to identify regulators of DNMT1 using immunoprecipitation assays; the only confirmed regulator of DNMT1, the E3 ubiquitin ligase UHRF1, was identified in a genetic screen, not an interaction screen. Null alleles of Uhrf1 phenocopy null alleles of Dnmt1 in mice . Recent studies suggest that UHRF1 acts through binding and ubiquitylation of histones and other proteins at the replication foci [32, 33] rather than by effects on DNMT1 expression.
These findings indicate that the steady-state level of DNMT1 protein in cycling cell populations is controlled at the level of transcription and that the interaction of DNMT1 and USP7 is unlikely to play a major role in DNMT1 homeostasis.
OY and THB designed the study, OY carried out the experimental procedures, OY and OT studied USP7-deficient mouse and human cells, and OY and THB wrote the paper. All authors read and approved the final manuscript.
We thank Mathieu Boulard for the gift of the IAP probe and for helpful discussions and comments on the manuscript, Zoha Shahabuddin for statistical analysis, and Lissette Delgado-Cruzada for help with LUMA assays.
The authors declare that they have no competing interests.
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All animal experimentation was conducted under protocols approved by the IACUC of Columbia University.
This study was supported by Grants from the NIH to WG and THB.
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