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Selective Targeting of TET Catalytic Domain Promotes Somatic Cell Reprogramming

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Selective Targeting of TET Catalytic Domain Promotes Somatic Cell Reprogramming Selective targeting of TET catalytic domain promotes somatic cell reprogramming Anup Kumar Singha, Bo Zhaoa, Xiuhua Liua, Xin Wanga, Hongzhi Lib, Hanjun Qinc, Xiwei Wuc, Yuelong Mad, David Horned, and Xiaochun Yua,1 aDepartment of Cancer Genetics & Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010; bComputational Therapeutic Core, City of Hope, Duarte, CA 91010; cIntegrative Genomics Core, City of Hope, Duarte, CA 91010; and dChemical Synthesis Core, City of Hope, Duarte, CA 91010 Edited by Peter A. Jones, Van Andel Institute, Grand Rapids, MI, and approved January 6, 2020 (received for review June 26, 2019) Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) (11, 12). Therefore, TET proteins may also have a profound effect oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine on somatic cell reprogramming, which is a process equivalent to (5hmC) marks on the genome. Each TET protein also interacts with the reversal of differentiation. Recent studies suggest that TET specific binding partners and partly plays their role independent of enzymes regulate this process of reprogramming by expressing catalytic activity. Although the basic role of TET enzymes is well a defined set of Yamanaka factors (Oct4, Sox2, Klf4, and Myc) in the established now, the molecular mechanism and specific contribution presence of TET proteins (13–15). However, the molecular mecha- of their catalytic and noncatalytic domains remain elusive. Here, by nism of TET enzymes in the context of somatic cell reprogramming combining in silico and biochemical screening strategy, we have still remains elusive. For instance, TET1 deficiency has been shown identified a small molecule compound, C35, as a first-in-class TET to promote somatic cell reprogramming in the presence of vitamin inhibitor that specifically blocks their catalytic activities. Using this C. In contrast, without vitamin C, TET1 alone can replace Oct4 inhibitor, we explored the enzymatic function of TET proteins and facilitate somatic cell reprogramming (16, 17). Moreover, it during somatic cell reprogramming. Interestingly, we found that has been reported that 5hmC may act as an independent epi- C35-mediated TET inactivation increased the efficiency of somatic genetic mark rather than just an intermediate during 5mC cell programming without affecting TET complexes. Using high- demethylation in the context of somatic cell reprogramming (7). throughput mRNA sequencing, we found that by targeting 5hmC Altogether, due to the redundant nature of TET enzymes and repressive marks in the promoter regions, C35-mediated TET lack of TET inhibitors, the molecular mechanism of TET en- CELL BIOLOGY inhibition activates the transcription of the BMP-SMAD-ID signal- zymes in somatic cells reprogramming is understudied. In par- ing pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important ticular, without a specific inhibitor, it is difficult to distinguish the tool for inducing somatic cell reprogramming, as well as for enzymatic functions of TET enzymes from their nonenzymatic dissecting the other biological functions of TET enzymatic activ- roles. Here, we have identified compound 35 (C35) as a first-in- ities without affecting their other nonenzymatic roles. class, highly potent, and cell-permeable TET inhibitor that rec- ognizes the catalytic core of TET enzymes and interferes with TET enzymes | somatic reprogramming | 5hmC | epigenetic reprogramming their enzymatic activities. Moreover, we found that C35 treatment NA methylation at the 5′-position of cytosine (5-methylcytosine; Significance D5mC) has emerged as one of the most common epigenetic modifications in genomic DNA, which plays an essential role in Ten-eleven translocation (TET) proteins mainly express during development, aging, and diseases (1–3). Recent studies have embryonic development and regulate tissue-specific differen- shown that the ten-eleven translocations (TET) family of methyl tiation. However, their specific role in somatic cell reprogram- cytosine dioxygenases (TET1, TET2, and TET3) can oxidize 5mC ming is elusive. Here, we report a small molecule inhibitor, C35, into various derivatives (4, 5). All three members of the TET that specifically targets TET catalytic domain and reduced 5- family share highly similar catalytic domains that bind DNA and hydroxymethylcytosine (5hmC) load on the genome. Our re- convert 5mC into 5-hydroxymethylcytosine (5hmC) in the pres- sults clearly indicate that this selective inhibition of TET cata- ence of cofactors including 2-oxoglutarate and Fe(II) (4). Further lytic activity reduced 5hmC marks genomewide and promotes oxidation of 5hmC by TETs generates 5-formylcytosine (5fC) and somatic cell reprogramming by inducing the BMP-SMAD-ID 5-carboxylcytosine (5caC) (5, 6). Both 5fC and 5caC are rela- downstream pathway. This finding will improve our current tively unstable on the chromatin (5, 6) and can be recognized by understanding about the differential role and contribution of thymine-DNA glycosylase (TDG) that mediates the replacement TET catalytic and noncatalytic domains in somatic cell reprog- of 5fC and 5caC with unmodified cytosine for the completion of ramming. C35 could also be a key tool for the scientific com- the active demethylation process (5). Hence, unlike 5fC or 5caC, munity to study the selective role of TET catalytic domain on 5hmC is a stable epigenetic mark in chromatin, suggesting that at various biological processes. least a portion of TET enzymes only catalyze the formation of – Author contributions: X.Y. designed research; A.K.S., B.Z., X.L., X. Wang, H.L., and Y.M. 5hmC, but not 5fC or 5caC (7 9). However, the biological func- performed research; A.K.S., H.L., H.Q., X. Wu, D.H., and X.Y. analyzed data; X.L. helped in tion of these stable 5hmC marks and their impacts on gene molecular docking; X. Wang performed high-performance liquid chromatography–high- transcription remain elusive. resolution mass spectrometry; and A.K.S. and X.Y. wrote the paper. Genetic evidence suggests that TET family members are func- The authors declare no competing interest. tionally redundant during embryonic development, and the loss of This article is a PNAS Direct Submission. any member of the TET family enzyme does not induce prenatal Published under the PNAS license. lethality in mice. However, the combined loss of TET1 and TET2 Data deposition: The data reported in this paper have been deposited in the Gene Ex- suppresses hematopoietic stem cell differentiation (10). Knocking pression Omnibus (GEO) database (accession no. GSE137282). out all three TET proteins arrests mouse embryogenesis at or 1To whom correspondence may be addressed. Email: [email protected]. before implantation and impairs the differentiation of embryonic This article contains supporting information online at https://www.pnas.org/lookup/suppl/ stem cells, suggesting that TET enzymes may play a key role in doi:10.1073/pnas.1910702117/-/DCSupplemental. tissue-specific differentiation by controlling key genes’ expression First published February 5, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1910702117 PNAS | February 18, 2020 | vol. 117 | no. 7 | 3621–3626 Downloaded by guest on September 26, 2021 clearly promotes somatic cell reprogramming. Mechanistic analyses suppress the enzymatic activity of TET2 during the screening; show that C35 treatment induced the expression of members of the however, their inhibitory effects were not as potent as that of C35. BMP-SMAD-ID axis that are known to play key roles in somatic Thus, C35 was selected for the subsequent studies. We have vali- cell reprogramming. dated the structural identity and purity of C35 using high-performance liquid chromatography–high-resolution mass spectrometry, 13C Results NMR, and 1H NMR (SI Appendix,Figs.S1–S3), and next Identification of C35 as a TET Inhibitor In Vitro. To identify novel performed a dose course experiment to examine the efficacy of TET inhibitors, a virtual ligand screening pipeline (Lvspipe) was C35 using the in vitro assays, and found that half-maximal in- employed (18). We selected the TET2 catalytic domain (PDB: hibitory concentration (IC50) of C35 against TET2 was 1.2 μM 4NM6) and performed a molecular modeling screening with the (Fig. 1D). Moreover, we purified the catalytic domain of human National Cancer Institute compound library that contains the TET1 and TET3 and examined the inhibitory effect of C35, using structures of ∼265,000 compounds. Based on the docking score the same in vitro assays. We found that similar to TET2, C35 was for their virtual interaction and other chemical properties, we also able to suppress the catalytic activities of TET1 and TET3 selected 40 top hits for additional in vitro biochemical screening with IC50 3.48 μMand2.31μM, respectively (SI Appendix,Fig.S4 (Fig. 1A). Purified catalytic domain of human TET2 was used for A and B). Thus, these results suggest that C35 is a general TET the in vitro screening in the presence of cofactors including 2- inhibitor that targets all members of the TET family. oxoglutarate (α-KG) and Fe(II). Using dot blotting assay with Next, we examined the inhibition mechanism of C35. Our mo- anti-5hmC antibody, we analyzed the conversion of 5mC into lecular modeling results indicate that C35 may partially occupy the 5hmC in the chemically synthesized DNA oligos. Among 40 DNA substrate binding sites and block the catalytic cage of TET2 different compounds, we found C35 to
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