The Oxidative Demethylase ALKBH3 Marks Hyperactive Gene Promoters in Human Cancer Cells
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The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Liefke, Robert, Indra M. Windhof-Jaidhauser, Jochen Gaedcke, Gabriela Salinas-Riester, Feizhen Wu, Michael Ghadimi, and Sebastian Dango. 2015. “The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells.” Genome Medicine 7 (1): 66. doi:10.1186/s13073-015-0180-0. http:// dx.doi.org/10.1186/s13073-015-0180-0. Published Version doi:10.1186/s13073-015-0180-0 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:17820928 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Liefke et al. Genome Medicine (2015) 7:66 DOI 10.1186/s13073-015-0180-0 RESEARCH Open Access The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells Robert Liefke2,3†, Indra M. Windhof-Jaidhauser1†, Jochen Gaedcke1, Gabriela Salinas-Riester4, Feizhen Wu5, Michael Ghadimi1 and Sebastian Dango1,2,3* Abstract Background: The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes upregulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood. Methods: To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion. Results: We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers, and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an upregulation of ALKBH3 non-bound inflammatory genes. Conclusions: The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression. Background acids, often requires repair to maintain genomic integ- Genomic DNA is continuously subjected to various rity. Alkylating agents are found ubiquitously in the en- harmful insults, such as UV light, ionizing radiation, or vironment, but DNA can also be alkylated as a natural nucleic-acid modifying compounds, resulting in thou- by-product of cellular metabolism [6, 7]. For example, sands of DNA alterations in each cell every day [1]. Such the universal methyl donor S-adenosylmethionine non- lesions can lead to DNA damage, which in turn favors enzymatically methylates DNA [8, 9]. Alkylating agents mutagenesis, carcinogenesis, inflammation, and aging preferentially attack single-stranded DNA (ssDNA) in [2–5]. Accordingly, cells have multiple mechanisms to the genome due to its higher accessibility [10–13], and reverse damaging DNA modifications. In particular, some DNA modifications such as 1-methyladenine DNA alkylation, a process of methylating specific nucleic (1-meA) and 3-methylcytosine (3-meC) are primarily generated in ssDNA, because these positions are shielded in double-stranded DNA (dsDNA) [6]. * Correspondence: [email protected] †Equal contributors DNA alkylation can be removed by base-excision 1University Medical Center, Department of General-, and Visceral Surgery, repair (BER), direct reversal by methylguanine meth- D-37075 Göttingen, Germany yltransferase (MGMT), and dealkylation via the AlkB 2Division of Newborn Medicine and Program in Epigenetics, Department of Medicine, Boston Children’s Hospital, Boston, MA 02115, USA family [6, 7]. The AlkB enzymes belong to a large Full list of author information is available at the end of the article family of non-heme Fe(II) and 2-oxoglutarate-dependent © 2015 Liefke et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liefke et al. Genome Medicine (2015) 7:66 Page 2 of 13 dioxygenases, which catalyze numerous biological reac- localization of ALKBH3 at transcription-related loci tions, such as proline hydroxylation and histone demeth- raises the possibility that ALKBH3 could have a role in ylation [14]. AlkB was original discovered in E. coli, suppressing transcription-associated DNA damage to where it demethylates 1-methyladenine (1meA) and 3- preserve the genomic integrity. methylcytosine (3meC) by oxidation of the N-linked methyl moiety. This reaction creates an unstable Methods methyl-iminium intermediate that spontaneously hy- Cell culture and viral transduction drolyzes into formaldehyde and the non-alkylated base U2OS, 293 T, NCI-H23, and PC3 cells were obtained [15–17]. In mammalians, at least nine ALKB family from the American Type Culture Collection (ATCC) members are known (ALKBH1-8 and FTO). DNA damage and maintained as previously described [24]. ShRNAs dealkylation reactions are mainly catalyzed by ALKBH2 constructs, preparation of viruses and cell transduction and ALKBH3 [16]. Notably, ALKBH2 preferentially have been described previously [24]. Cells infected with demethylates dsDNA while ALKBH3 demethylates lentiviral shRNAs were selected after infection with ssDNA and RNA substrates, modified by 3-meC or 1-meA puromycin (1 μg/mL) for at least 48 h. [16, 18, 19]. Since these modifications are predominantly generated in ssDNA and RNA, it has been proposed that Antibodies the ALKBH3 repair function could be linked to transcrip- Rabbit anti-ALKBH3 antibodies were obtained from tion [6, 20]. Incomplete removal of DNA alkylation leads Millipore (Catalog #09-882). to DNA damage, resulting in cell cycle arrest, inflammation and apoptosis [3–5, 21]. Induction of DNA alkylation by Immunofluorescence (IF) chemotherapeutic agents is a common strategy in cancer U2OS and PC3 cells were used for IF and were main- treatment to prevent cancer cells from dividing and prolif- tained as described previously [24]. The cells were in- erating [22]. Enzymes that facilitate DNA alkylation dam- fected with the indicated lentiviral shRNAs and plated age repair, such as ALKBH2 and ALKBH3, can contribute onto coverslips. Cells were then fixed with PBS (ph 7.4) to resistance to this treatment and insights into their mo- containing 3.2 % paraform for 20 min, washed exten- lecular function could provide the basis for developing sively with IF wash buffer (1X PBS containing 0.5 % NP- more efficient cancer therapies [23]. 40 and 0.02 % NaN3), then incubated with blocking Recently, we described the cooperativity of ALKBH3 buffer (IF wash buffer with 10 % fetal bovine serum), and the ASCC3 DNA helicase complex to promote and finally stained with anti-rabbit-ALKBH3 (Millipore) DNA alkylation damage repair in various cancer cells. diluted in blocking buffer. Secondary antibodies (goat ALKBH3 knockdown causes elevated levels of 3-meC ac- anti-rabbit Alexa Fluor 488) were from Millipore. companied by increased DNA damage response (DDR) and reduced cell proliferation [24]. However, the mecha- RNAi experiments, microarray analysis, and qRT-PCR nisms of in vivo genomic targeting of ALKBH3 are not yet Lentiviral shRNA against ALKBH3, ALKBH2, and GFP fully understood. as control were used as described before [24]. Briefly, Herein, using chromatin immunoprecipitation experi- lentiviral production was carried out in 293 T cells and ments followed by massively parallel sequencing analysis target cells were infected for 48 h followed by selection (ChIP-seq) we find that in PC3 prostate cancer cells with puromycin for 48 h or 96 h. RNA extracted from ALKBH3 binding is enriched at transcription associated ALKBH3 knockdown and control cells were sent to genomic loci, where ssDNA is accessible. Specifically, we Transcriptome Analysis Laboratory (TAL, University find ALKBH3 bound at active gene promoters, en- Medical Center, Göttingen) for expression analysis. hancers, and regions with putative quadruplex DNA. Microarrays were done using the ‘Low RNA Input linear Unexpectedly, ALKBH3 binds strongly to the initiation Amplification Kit Plus, One Color’ protocol (Agilent sites of some particularly highly expressed gene pro- Technologies, Cat. No.: 5188–5339) and the Agilent moters. Interestingly, these promoters are bound by an RNA Spike-In Kit for One color (Agilent Technologies,