Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader–Willi syndrome

Prader–Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11–q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642—two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)—activated the maternal (m) copy
of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pDS−U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pS−U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.

PWS is clinically characterized by neonatal hypotonia and failure to thrive, childhood-onset obesity, intellectual disability and increased risk for psychosis in adults1. Although paternal deficiency of the 15q11–q13 chromosomal region is well documented as the etiology of PWS, the precise molecular basis underlying these clinical features remains elusive. Paternally expressed genes within 15q11–q13, includ- ing SNRPN, MAGEL2, NDN and MKRN3, and noncoding SnoRNA clusters of SNORD115 (HBII-52) and SNORD116 (HBII-85), are con- trolled by a regulatory element defined as an imprinting center (PWS- IC)2. Among these genes, the SnoRNA cluster SNORD116, located between SNRPN and UBE3A, plays a critical part in PWS etiology, as indicated by genomic copy-number variant (CNV) analyses3–6, and the specific role of MAGEL2 in PWS remains a subject of debate owing to conflicting findings in humans3,7–9.
SNORD116 is processed from its host transcript, a long noncoding RNA that is thought to initiate at the PWS-IC10. Human SNORD116 and mouse Snord116, including host transcripts, are highly conserved in their genomic organization and imprinted expression patterns10–12; yet the mechanism underlying the imprinted expressions of SNPRN and SNORD116 is still unclear. DNA methylation and histone modifi- cation are common mechanisms thought to be implicated in genomic imprinting. The differential methylation of CpG islands in the PWS-IC is consistent with the paternal activation of the genes, i.e., they are fully methylated on the maternal chromosome but unmethylated on the paternal chromosome13.

However, histone modifications such as the acetylation of histone H3 lysine 4 (H3K4) and the methylation of histone H3 lysine 9 (H3K9) also exhibit allele-specific patterns in the PWS-IC14,15. Although histone modification is expected for transcriptional regulation, its role in the regulation of imprinted genes is less clear and has been viewed as an event secondary to, or as a substitute for, DNA methylation. DNA methylation inhibitors can activate the expression of the maternal-originated SNRPN in vitro16, but this has not been reported in vivo. In addition, the inactivation of histone H3K9 methyltransferase G9a in mouse embryonic stem (ES) cells in vitro can induce the biallelic expression of Snprn that occurs with reduced DNA methylation17. Of note, DNA methylation of Snrpn is not affected in embryonic day 9.5 (E9.5) G9a−/− mouse embryos17,18, yet the allele-specific expression of Snrpn in the absence of G9a is not known in vivo. Thus, we envisioned that epigenetic manipulation of the PWS imprinting domain could enable the maternal chromosome to express PWS-associated genes that normally show paternal-specific expression, and thus provide a therapeutic strategy for PWS.

RESULTS
Identification of G9a inhibitors that activate candidate PWS-associated genes by a high-content screenOur primary objective was to identify small molecules that are capable of activating the expression of SNORD116 from the maternal chromo- some, and so might offer therapeutic benefit for PWS. However, it was not feasible to design a screening for noncoding RNA. SNRPN (mouse Snrpn), however, is a protein-coding gene that is expressed paternally,but repressed maternally, in all tissues in human and mouse19. The allele-specific expression of human SNRPN is regulated by the PWS-IC, which also controls the expression of host transcripts for SnoRNAs, including the SNORD116 cluster between SNRPN and UBE3A20. Thus, we decided to use the Snrpn-EGFP fusion protein (hereafter, S-EGFP) as a marker for a HCS campaign. We reasoned that small molecules that can activate S-EGFP might also be effective at activating the host transcript of Snord116. Accordingly, we established mouse embryonic fibroblasts (MEFs) from mice carrying S-EGFP inherited either maternally (mS-EGFP/p+) or paternally (m+/pS-EGFP), in which EGFP is inserted after exon 2 of the Snurf–Snrpn bicistronic transcript21. We confirmed that S-EGFP was expressed in m+/pS-EGFP and repressed in mS-EGFP/p+ MEFs (Supplementary Fig. 1a). The MEFs of mS-EGFP/p+ were then subjected to a HCS using a protocol that we described previously22 (Fig. 1a). We performed the screen in quadruplicate, using 13 small-molecule libraries from multiple sources, including three random epigenetic-library collections (10 M in 0.2% DMSO; Fig. 1b and Supplementary Table 1).

We chose these libraries to ensure chemical diversity and pharmacological and bio- logical activity. After employing an initial arbitrary cut-off of 125% (100% indicates basal fluorescence in the vehicle-treated MEFs), out of 9,157 compounds (Fig. 1b), we identified 32 potentially active compounds from the primary screen (Supplementary Fig. 1b and Supplementary Table 2). Two of these compounds, UNC0638 and UNC0642, were validated and shown to be active in orthogonal assays of immunocytochemistry (Fig. 1c), concentration responses (Fig. 1d) and quantitative reverse transcription PCR (RT–qPCR) (Fig. 1e).Both UN0638 and UNC0642 have been characterized as G9a- selective inhibitors that bind and block the catalytic domain of G9a23,24. Through an extended screening of 23 UNC0638 and UNC0642 analogs, we subsequently identified two additional com- pounds: UNC617 (Supplementary Fig. 2) and UNC618 (ref. 25) that could also activate the expression of S-EGFP in mS-EGFP/p+ MEFs (Fig. 1c–e). UNC0638, UNC0642 and UNC617 displayed similar potencies in concentration-response studies (Fig. 1d; half-maximal effective concentration (EC50) = 1.6 M for UNC0638, 2.7 M forUNC0642 and 2.1 M for UNC617). The estimated maximal effective- ness (Emax) was similar for these three compounds, whereas UNC618 was effective only at 30 M. Next, we performed RT–qPCR to measure the changes in mRNA of S-EGFP. These compounds upregulated the mRNA of S-EGFP to an extent comparable to or greater than that induced by 10 M of 5-aza deoxycytidine (5-Aza-dC), an inhibi- tor of DNA methyltransferases (DNMTs) (Fig. 1e).

Because other allele-specific histone modifications, such as acetylation, occur in the PWS-IC16, we next examined whether the modulation of other classes of histone-modifying enzymes can activate S-EGFP. However, as summarized in Supplementary Table 3, we did not observe activation of S-EGFP in the presence of different classes of modula- tors. Notably, BIX01294 (ref. 26), the first reported G9a inhibitor, which is less potent than UNC0638 and UNC0642, did not have a sub- stantial effect on the activation of S-EGFP (Supplementary Table 3). These data suggest that the effects of our active compounds are rela- tively specific and probably result from targeting specific histone methyltransferases.Next, we tested whether these drugs could derepress the maternal genes in a patient-driven cell model of PWS: a human skin fibroblast cell line containing a typical large 5–6-Mb deletion of the paternalcopy of the 15q11–q13 region (Fig. 2a). Because imprinting of SNRPN is known to be ubiquitous19, the G9a-inhibitor effect on its activation is expected to be representative of all tissues and cell types. Using the indicated treatment scheme (Fig. 2b), we compared the effectiveness of four identified compounds from the HCS, including a control, 5-Aza-dC. All of them displayed apparent activation of SNRPN mRNA expression from the maternal chromosome (Fig. 2c). However, only UNC0638 and UNC0642 were effective for SNORD116 and the putative host transcripts for SNORD116 (116HG) and SNORD115 (115HG). For 116HG, multiple bands were seen in the drug-treated cells, and these products were further examined through subsequent Sanger sequencing. As indicated, some of these products were mapped to the region of 116HG (Fig. 2c), and the majority of the sequences were not specific or partially matched to the long-terminal-repeat sequence, a known sequence that requires G9a for its repression.

The UNC0638 and UNC0642 treatments also reactivated the expres- sion of NDN, which is 1 Mb proximal to PWS-IC. We were not able to determine the expressions of MAGEL2 and SNORD115, despite the activation of 115HG, because they are not normally expressed in skin fibroblasts28–30.We chose UNC0638 for follow-up cell-based studies because of its high potency and selectivity, low toxicity and thoroughly characterized cellular activity23. UNC0638 treatment (1–4 M) effectively activated SNRPN and SNORD116 transcripts, as assessed by RT–PCR (Fig. 2d), with minimal cytotoxicity (Supplementary Fig. 3). PWS fibroblasts treated with 4 M of UNC0638 expressed approximately 30% of control SNRPN protein levels (Fig. 2e and Supplementary Fig. 4).Taken together, these expression analyses strongly indicate that UNC0638 and UNC0642 are capable of activating the maternal copy of the paternally expressed genes from the PWS-associated region.Using the m+/pS−U mouse model of PWS31, we next examined the effects of UNC0642 in vivo. Neonatal m+/pS−U mice display perinatal lethality and poor growth31 that resembles the failure-to-thrive fea- ture of individuals with PWS during the first year of life1. We chose UNC0642 because it has not only high potency and selectivity for G9a in biochemical and cellular assays, but also excellent pharmacokinetic (PK) properties, including CNS penetration superior to UNC0638 (ref. 24).

A single intraperitoneal (i.p.) injection dose of 5 mg/kg of UNC0642 is sufficient to inhibit G9a activity in adult mice24. We administered the G9a inhibitor UNC0642 in a blinded and rand- omized fashion to mouse genotypes between postnatal day 7 (P7) and P12, because most m+/pS−U pups died before weaning. For neonatal mice, we used a lower dosage regimen of 2.5-mg/kg i.p. injections for 5 consecutive days (Fig. 3a). The UNC0642 treatment was well tol- erated by both wild-type (WT) and m+/pS−U pups and significantly attenuated lethality in m+/pS−U mice as compared to the untreated m+/pS−U group (Fig. 3b). The difference in the survival rates of PWS pups was most notable during the first week after drug administration and diminished over time. Six UNC0642-treated m+/pS−U pups sur- vived to >P90 (15%; n = 40), and they had normal physical appearance (Fig. 3c) and activity in their home cages. Body-weight measurementsrevealed that there was a significant improvement of growth between P10 and P19 in treated m+/pS-U pups (Fig. 3d). These results indicate the—albeit partial—rescue of lethality and growth-delay phenotypes of the PWS mouse model, and hence the potential of such treatment for humans.To assess the potential toxicity associated with UNC0642 treatment, we monitored body weight in WT groups. Notably, loss of body weight, a sign of general health deficiency, was not observed in WT mice treated with UNC0642 (Fig. 3e).

We also performed a general health and neurological screening in a blinded fashion, and it did not reveal any substantial abnormalities (Supplementary Table 4). In additional toxicity tests, we did not include vehicle- treated PWS mice because of the small sample size. Despite our breed- ing effort that produced a total number of 60 m+/pS−U pups, only two vehicle-treated m+/pS−U mice survived to P90. In hematologicalanalysis, the measurements of treated m+/pS−U and WT mice were within normal ranges, as measured by liver and kidney func- tions as well as normal lipid and protein metabolism, which are indicative of normal health conditions (Supplementary Table 5). Histopathological analyses also did not reveal any abnormalities associated with UNC0642 treatment in the brain, liver, kidney, lung and heart from mice at P90, both in m+/pS−U and WT mice (Supplementary Fig. 5).We next assessed RNA and protein expression in m+/pS−Umice at around P14 following UNC0642 treatment (Fig. 4a). The expression of Snrpn and Snord116 was readily detectable in the brain and liver—two organs relevant to the pathogenesis of PWS—of UNC0642 treated m+/pS−U mice by RT–PCR (Fig. 4b) and quantitative reverse transcription PCR (RT–qPCR) (Fig. 4c), whereas vehicle-treated m+/pS−U mice had no detectable transcripts(Fig. 4b,c). We also examined whether this activation affects the maternal expression of Ube3a because its antisense transcript (Ube3a-ATS), which has an essential role in repressing the paternal copy of Ube3a in the brain, is also only paternally expressed32,33. Notably, the expression of Ube3a-ATS was not affected in the brain (Fig. 4c).

Similarly, the Ube3a protein level was not changed in whole brain (Fig. 4d), or spe- cifically, in the cerebellum, where the maternal-specific Ube3a tran- script is predominantly expressed34 (Supplementary Fig. 6). We also accessed the treatment effect in adulthood using the mS-EGFP/p+ mouse model (Fig. 4e–g). Treatment of 6-week-old mice exerted a long- lasting effect, as shown by the maternal expression of Snrpn–EGFP at 0 (Fig. 4f), 1, 4 and 12 weeks (Fig. 4g) after the final dose of UNC0642. However, it is worth noting that expression levels at 12 weeks were significantly lower than those at 4 weeks (P = 0.03). Therefore, these results demonstrate the efficacy of UNC0642 treat- ment in vivo for the mouse model of PWS and provide sufficient proof of principle to consider evaluating such a therapeutic intervention, targeted at the molecular etiology of PWS.We next investigated the underlying mechanism for the activation of the maternal chromosome 15q11–q13 by UNC0638 and UNC0642. The PWS-IC is methylated on the maternal chromosome, but unmeth- ylated on the paternal chromosome2. This allele-specific methylation is thought to implicate the imprinted regulation of candidate PWS- associated genes14,16. Given that G9a is also known to modulate DNA methylation via the ankyrin-repeat domain (ANK) but not the cata- lytic domain of G9A35,36, we first examined the differential methyla- tion to see whether the activation of the maternal genes by the G9a inhibitors occurs directly or through the loss of DNA modification. As expected, 5-Aza-dC significantly decreased DNA methylation of the PWS-IC as compared to the vehicle-treated group when examined by a bisulfite genomic-sequencing method (P < 0.05).

By contrast, the UNC0638 and UNC0642 did not significantly alter DNA meth- ylation of the PWS-IC in human PWS cells or in m+/pS−U mice, respectively, as analyzed by the bisulfite genomic-sequencing method(Fig. 5a), which is in agreement with the previous report showing that UNC0638 does not alter global DNA methylation23. These data suggest that maternal activation is partially independent from the loss of DNA methylation, even though a dose-dependent hypomethyla- tion of long terminal repeats (LTRs) for individual genomic loci was observed in UNC0638-treated cells23.We next examined whether these inhibitors affected the H3K9 methylation pattern. Both H3K9me2 (dimethylation of H3K9)15,17 and H3K9me3 (trimethylation of H3K9)37 are associated with the maternal chromosome in the PWS region. We verified differential histone modifications of the PWS-IC by a chromatin immunopre- cipitation (ChIP) assay, in which H3K9me2 and H3ac (acetylation of H3) were enriched at the maternal or paternal PWS-IC, respectively (Supplementary Fig. 7). We also noted that treatment with 5-Aza-dC also reduced H3K9me2 in the PWS-IC of the maternal chromosome as compared to the vehicle-treated group (Supplementary Fig. 7). The suppression of DNA methylation thus seems to impair H3K9 methylation at the PWS-IC, possibly owing to the loss of interaction between DNMTs and G9a35,36. Using the MAGE-A2 promoter38 and a centromere sequence (CEN)15 as controls, UNC0638 drastically reduced the levels of H3K9me2 and H3K9me3 in the PWS-IC and SNORD116 regions (Fig. 5b,c). H3K9me2, but not H3K9me3, was enriched at the PWS-IC, and treatment with UNC0638 reduced H3K9me2 as compared to the untreated control cells in PWS-IC (Fig. 5b,c).

The UNC0638 treatment also reduced H3K9me2 of other examined sites along the PWS region, including the promoter of NDN (Supplementary Fig. 8). At the region of the host transcript of SNORD116, both H3K9me2 and H3K9me3 were enriched, and UNC0638 treatment reduced H3K9me2 (Fig. 5b,c) and H3K9me3 (Fig. 5b,d) as compared to the untreated controls .Although G9a catalyzes primarily mono- and dimethylation reactions on H3K9 by its Su(var)3-9 and enhancer of zeste (SET) domain, it can contribute to the trimethylation of H3K9 in individual loci via an undefined biochemical mechanism39,40. This undefined regulation might account for the changes in H3K9me3 at the SNORD116 region. Despite the reduction of H3K9me2 at the MAGE-A2 promoter, we did not see the transcriptional activation of MAGE-A2 (Supplementary Fig. 9a). This is probably because MAGE-A2 activation by G9a inhibi- tors is cell type– and treatment condition–dependent23,26. We also tested the effects of the G9a inhibitor on the other imprinted loci, including maternally expressed CDKN1C, and paternally expressed IGF2 and PEG10. We found that the expression of only the pater- nally expressed PEG10 was modestly upregulated by the G9a inhibitor (Supplementary Fig. 9b). This suggests that the inhibition of G9a affects gene expression in a locus-selective manner, and this is con- sistent with the study reporting that only 1 of 16 imprinted loci are selectively affected in G9a−/− embryos18.We next performed G9a chromatin immunoprecipitation (ChIP) analysis. G9a recognizes H3K9me2 through its ANK domains, which might amplify the spreading of H3K9me2 (refs. 41,42).

The associations of G9a with the PWS-IC and SNORD116 region were not significantly affected (Fig. 5e), which indicates that UNC0638 does not impair the binding of G9a to chromatin. Given that DNA methylation did not change in the presence of the G9a inhibitors, it might further suggest that the intact protein–protein interaction between ANK and DNMTs can still be capable of modulating DNA methylation. This idea is supported by previous reports showing that the ANK domain, but not the catalytic domain, of G9a is essential to maintaining the DNA methylation of imprinted genes35,43.G9a-inhibitor treatment led to more open chromatin in the PWS imprinted domainH3K9me2 facilitates heterochromatin formation to regulate transcrip- tion38,40. We thus investigated whether the reduction of H3K9 meth- ylation could result in more open chromatin across the imprinted domain. Quantitative PCR of genomic DNA following in situ nuclease digestion was performed to measure chromatin accessibility (Fig. 6a), using a previously described protocol44. We used the following con- trols in this study: the constitutively expressed GAPDH, which was highly susceptible to nuclease digestion, and constitutively silent rho- dopsin, which displayed minimal chromatin accessibility, regardless of UNC0638 treatment (Supplementary Fig. 10). As a result of the treatment with UNC0638, the target regions across the imprinted domains, including the SNRPN and SNORD116, were more open and accessible than vehicle-treated controls (Fig. 6b). The effect of UNC0638 and UNC0642 seemed to be bidirectional in reference to the PWS-IC in the PWS domain.Taken together, these results suggest that the reduction of H3K9 methylation, but not DNA demethylation of PWS-IC, by the UNC0638 and UNC0642 treatment leads to more open chromatin, which, in turn, activates candidate PWS-associated genes from the maternal chromosome (Fig. 6c).

DISCUSSION
We discovered that the G9a inhibitors UNC0638 and UNC0642, identified from an unbiased small-molecule HCS, activate the candidate PWS-associated genes from the maternal chromosome both in human PWS patient-derived cells and in a mouse model of PWS. Treatment with UNC0642 afforded a clear therapeutic ben- efit for PWS-related phenotypes, including perinatal lethality and poor growth, which resemble the common clinical features of fail- ure to thrive in individuals with PWS during the first year of life31. Further studies are necessary to determine whether G9a inhibitors might offer therapeutic benefit to other major clinical problems of PWS, such as obesity, hyperphagia and behavioral impairment, that occur in childhood or later1, when appropriate animal models of PWS become available. We also show that UNC0642 treatment does not affect the expres- sion of Ube3a, a maternally expressed gene whose loss causes Angelman syndrome (AS). The activation of PWS-associated genes on the maternal chromosome raises a concern because it may acti- vate Ube3a antisense RNA (Ube3a-ATS), which normally represses paternal Ube3a expression32,33 but is not expressed from the maternal chromosome45. It is unclear how the derepression of the PWS-associated genes Snrpn and Snord116 occurs without affecting the expression of Ube3a-ATS. The generation and the processing of host transcripts from the interval between PWS-IC and Ube3a are not well understood. In contrast to the current notion of a long transcript IC-SNURF-SNRPN10, we speculate that the expressions of Snord116 host transcript and Ube3a-ATS are regulated differently. A recent study in human tissues from healthy individuals found potential transcription start sites (TSSs) within the interval between PWS-IC and UBE3A (ref. 46): one between SNORD116 and SNORD115 clusters and another between SNORD115 and the 3 end of UBE3A.

The large host transcript from the PWS-IC, which overlaps with the SNRPN promoter, might stop before these additional TSSs, and UBE3A-ATS might be initiated from one of potential TSSs, probably the one close to the 3 end of UBE3A. It seems that our G9a-inhibitor treatment derepresses the PWS-IC overlapping with Snrpn promoter, but not the TSS of Ube3a-ATS on the maternal chromosome. The continuous distribution of H3K9me2 along the PWS domain does not extend to the distal region47, which then makes the TSS of Ube3a-ATS not targetable by the G9a inhibitor. Another possibility is that the effect of the G9a inhibitor might become weaker at the farther end distal to the PWS-IC. It is not well understood how the functions of histone methylation and DNA methylation are linked for the repression of the PWS-associ- ated imprinting domain in vivo. A previous genetic study showed that the PWS-IC was demethylated in G9a-deficient embryonic stem (ES) cells, whereas it was not affected in G9a-deficient mouse embryos17. Unfortunately, the expressions of PWS-associated genes have not been examined specifically in G9a-deficient embryos (which died at E9.5)38, presumably owing to technical difficulties associated with determining their allele-specific expression in embryonic tissue. We demonstrate that the repressed SNRPN and SNORD116 are activated by the pharmacological inhibition of G9a, and that the reactivation occurred without any alteration of DNA methylation (5-methylcyto- sine, 5mC) of the PWS-IC both in vitro and in vivo.

It should be noted that the possibility of modifications other than 5mC in PWS-IC being affected by the G9a inhibitor cannot be ruled out because the bisulfite method used for our DNA-methylation analysis cannot distinguish between 5mC and 5-hydroxymethylcytosine (5hmC), or between cytocine (C) and 5-carboxycytosine (5CaC)48–50. Nevertheless, the finding provides novel insights into the regulation of imprinting, whereby H3K9 methylation has a decisive role in the repression of PWS-associated genes on the maternal chromosome. We propose a chromatin-spreading model for the maternal activa- tion of PWS-associated genes, in which a reduction of H3K9 methyla- tion is sufficient for altering the chromatin state so that it becomes permissive to transcriptional activation. Previous genome-wide chro- matin profiling has revealed the regions of large, organized chromatin K9 modification (LOCK), including one from MKRN3 to the 3 end of UBE3A in the PWS-associated imprinted domain47. We underscore in the model that reduced H3K9me2 in the PWS-IC initiates the spread of open chromatin along the PWS-associated imprinted region. However, our data do not rule out another possibility, wherein the reduced H3K9 methylation in the individual loci across the imprinted domain could also contribute to more open chromatin after G9a- inhibitor treatment.

Our findings provide a proof of principle to develop small-molecule- based epigenetic therapy for human PWS (Fig. 6d). From a transla- tional perspective, the G9a inhibitor UNC0642 improves the life span and weight gain of m+/pS−U pups, produces long-lasting activation of PWS-associated genes, is apparently well tolerated and produces no notable acute toxicity, and does not interfere with the expression of the AS-associated Ube3a gene. For the development of new drug therapy, potential off-target effects associated with epigenetic drugs raise a gen- eral safety concern. The well-tolerated response to UNC0642, however, suggests that each case may be evaluated individually with careful con- sideration to the dose, duration, route and timing of drug delivery. The tolerability of the US Federal and Drug Administration–approved DNA-methylation inhibitor, observed in patients with myelodysplastic syndrome, is one example of the safety of an epigenetic drug51,52. It is also noteworthy that, in reported human cases, the disruption of multiple imprinted loci as a result of mutations in zinc-finger protein 57 (ZFP57), a transcriptional factor, result in only a relatively rare but mild condition of transient neonatal diabetes53,54, and suggests a more complex possibility for evaluating the broad effects of epigenetic drugs. Our study provides a crucial step toward the development of a specific molecular therapy for human PWS. On the basis of these promising results, comprehensive evaluation of the efficacy and tolerability of G9a inhibitors in preclinical studies is warranted to fully explore their therapeutic potential for treating PWS.