The oxidative demethylase ALKBH 3 marks hyperactive gene promoters in human cancer cells

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 Genomic DNA is continuously subjected to various harmful insults, such as UV light, ionizing radiation, or nucleic-acid modifying compounds, resulting in thousands of DNA alterations in each cell every day [1]. Such lesions can lead to DNA damage, which in turn favors mutagenesis, carcinogenesis, inflammation, and aging [2–5]. Accordingly, cells have multiple mechanisms to reverse damaging DNA modifications. In particular, DNA alkylation, a process of methylating specific nucleic * Correspondence: sebastian.dango@med.uni-goettingen.de Equal contributors University Medical Center, Department of General-, and Visceral Surgery, D-37075 Göttingen, Germany Division of Newborn Medicine and Program in Epigenetics, Department of Medicine, Boston Children’s Hospital, Boston, MA 02115, USA Full list of author information is available at the end of the article © 2015 Liefke et al. This is an Open Access art (http://creativecommons.org/licenses/by/4.0), provided the original work is properly credited creativecommons.org/publicdomain/zero/1.0/ acids, often requires repair to maintain genomic integrity. Alkylating agents are found ubiquitously in the environment, but DNA can also be alkylated as a natural by-product of cellular metabolism [6, 7]. For example, the universal methyl donor S-adenosylmethionine nonenzymatically methylates DNA [8, 9]. Alkylating agents preferentially attack single-stranded DNA (ssDNA) in the genome due to its higher accessibility [10–13], and some DNA modifications such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) are primarily generated in ssDNA, because these positions are shielded in double-stranded DNA (dsDNA) [6]. DNA alkylation can be removed by base-excision repair (BER), direct reversal by methylguanine methyltransferase (MGMT), and dealkylation via the AlkB family [6, 7]. The AlkB enzymes belong to a large family of non-heme Fe(II) and 2-oxoglutarate-dependent icle distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, . The Creative Commons Public Domain Dedication waiver (http:// ) 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 reactions, such as proline hydroxylation and histone demethylation [14]. AlkB was original discovered in E. coli, where it demethylates 1-methyladenine (1meA) and 3methylcytosine (3meC) by oxidation of the N-linked methyl moiety. This reaction creates an unstable methyl-iminium intermediate that spontaneously hydrolyzes into formaldehyde and the non-alkylated base [15–17]. In mammalians, at least nine ALKB family members are known (ALKBH1-8 and FTO). DNA damage dealkylation reactions are mainly catalyzed by ALKBH2 and ALKBH3 [16]. Notably, ALKBH2 preferentially demethylates dsDNA while ALKBH3 demethylates ssDNA and RNA substrates, modified by 3-meC or 1-meA [16, 18, 19]. Since these modifications are predominantly generated in ssDNA and RNA, it has been proposed that the ALKBH3 repair function could be linked to transcription [6, 20]. Incomplete removal of DNA alkylation leads to DNA damage, resulting in cell cycle arrest, inflammation and apoptosis [3–5, 21]. Induction of DNA alkylation by chemotherapeutic agents is a common strategy in cancer treatment to prevent cancer cells from dividing and proliferating [22]. Enzymes that facilitate DNA alkylation damage repair, such as ALKBH2 and ALKBH3, can contribute to resistance to this treatment and insights into their molecular function could provide the basis for developing more efficient cancer therapies [23]. Recently, we described the cooperativity of ALKBH3 and the ASCC3 DNA helicase complex to promote DNA alkylation damage repair in various cancer cells. ALKBH3 knockdown causes elevated levels of 3-meC accompanied by increased DNA damage response (DDR) and reduced cell proliferation [24]. However, the mechanisms of in vivo genomic targeting of ALKBH3 are not yet fully understood. Herein, using chromatin immunoprecipitation experiments followed by massively parallel sequencing analysis (ChIP-seq) we find that in PC3 prostate cancer cells ALKBH3 binding is enriched at transcription associated genomic loci, where ssDNA is accessible. Specifically, we find ALKBH3 bound at active gene promoters, enhancers, and regions with putative quadruplex DNA. Unexpectedly, ALKBH3 binds strongly to the initiation sites of some particularly highly expressed gene promoters. Interestingly, these promoters are bound by an unusually large number of transcriptional regulators, indicating a highly regulated ‘hyperactive’ promoter class. However, we find that loss of ALKBH3 does not directly affect expression of ALKBH3 occupied genes, suggesting a transcription unrelated function of ALKBH3. Instead, upon ALKBH3 knockdown we observe an increased expression of genes involved in inflammatory pathways, which could be a downstream effect of elevated DNA damage after ALKBH3 depletion [24, 25]. The genomic localization of ALKBH3 at transcription-related loci raises the possibility that ALKBH3 could have a role in suppressing transcription-associated DNA damage to preserve the genomic integrity.