Genome-Wide Localization of the Polyphenol Quercetin in Human Monocytes Dana Atrahimovich1,2, Avraham O

Atrahimovich et al. BMC Genomics (2019) 20:606 https://doi.org/10.1186/s12864-019-5966-9 RESEARCHARTICLE Open Access Genome-wide localization of the polyphenol quercetin in human monocytes Dana Atrahimovich1,2, Avraham O. Samson2, Yifthah Barsheshet2, Jacob Vaya1,3, Soliman Khatib1,3 and Eli Reuveni2* Abstract Background: Quercetin is a polyphenol of great interest given its antioxidant activity and involvement in the immune response. Although quercetin has been well studied at the molecular level as a gene regulator and an activator of specific cellular pathways,notmuchattentionhasbeengiventoitsmechanismofactionat the genome-wide level. The present study aims to characterize quercetin’s interaction with cellular DNA and to show its subsequent effect on downstream transcription. Results: Two massive parallel DNA-sequencing technologies were used: Chem-seq and RNA-seq. We demonstrate that upon binding to DNA or genome-associated proteins, quercetin acts as a cis-regulatory transcription factor for the expression of genes that are involved in the cell cycle, differentiation and development. Conclusions: Such findings could provide new and important insights into the mechanisms by which the dietary polyphenol quercetin influences cellular functions. Keywords: Polyphenol, Quercetin, Chem-seq, RNA-seq, Anticancer Background also interact with, and modulate the endogenous activ- Quercetin is a major polyphenol, known for its anti-in- ities of estrogen receptors, thereby slowing or even pre- flammatory, neuroprotective, cardioprotective, and che- venting the development of breast and ovarian cancers mopreventive activity [1, 2]. Quercetin is ubiquitous in [8, 9]. In addition, polyphenols have been found to inter- fruit, vegetables, teas and wines, as well as in dietary act with various transcription factors and to regulate supplements. For many years, quercetin’s mode of action cell-cycle arrest, apoptosis and survival [10]. Polyphenols was assumed to stem from its antioxidant properties— have been shown to interact with enzymes (e.g., hydro- donating electrons or chelating transition metals. How- lases, oxidases, kinases), altering their structure and ac- ever, antioxidant activity is not the sole explanation for tivity [11, 12]. Similarly, they have been found to act as quercetin’s cellular effects in vivo, since it is poorly ligands of nuclear receptors, modulating homeostasis absorbed, extensively metabolized, and its blood concen- [3]. Recent studies in our laboratory have found that trations barely reach the level needed for effective anti- polyphenols bind specifically to plasma proteins and oxidant activity [3]. Recent studies have suggested that lipoprotein particles and modify the lipoprotein’s phys- polyphenols’ mode of action is mediated by different, ical and biological structure. In another study, we found not yet fully explored mechanisms, such as interaction that quercetin binds to an allosteric site of the serum en- with proteins and nucleic acids [4]. Polyphenols in gen- zyme paraoxonase 1, affecting its function and biology eral, and quercetin in particular, readily penetrate cellu- [11]. Although polyphenols are known to regulate gene lar and nuclear membranes and accumulate in the cell expression and modulate signal-transduction pathways, nucleus [5]. Polyphenols have been shown to interact se- the precise mechanisms remain unclear [13]. Experi- lectively with different components of protein kinases ments in the last 3 years have suggested that polyphenols and alter their phosphorylation state, thus regulating bind to specific DNA-sequence motifs; for example, multiple cell-signaling pathways [6–8]. Polyphenols can quercetin interacts with the dodecamer duplex sequence, whose structure has been solved (PDB ID: 1BNA) [14]. * Correspondence: [email protected] Recently, the structure of quercetin complexed with c- 2Faculty of Medicine in the Galilee, Bar-Ilan University, 1311502 Safed, Israel Full list of author information is available at the end of the article myc G-quadruplex DNA was solved by nuclear magnetic © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Atrahimovich et al. BMC Genomics (2019) 20:606 Page 2 of 9 resonance (NMR) spectroscopy [15]. While the interac- Figure S1b), and found to correspond to the expected tions of some polyphenols, such as quercetin, resvera- mass. trol, genistein and curcumin with DNA are known, the mode of binding, the precise location of polyphenol- Localization of quercetin genomic binding sites using binding sites on the DNA, and their function at the Chem-seq genome level have not been investigated [16]. To identify the genome-wide binding activity of quer- Today, next generation sequencing (NGS) technologies cetin in human monocyte cell line THP 1, we used the can provide the complete genome of an organism fol- Chem-seq technique schematically described in Fig. 2b lowing an established laboratory protocol. Moreover, it to determine whether quercetin is associated with regu- is possible to extract DNA from specific regions by cell latory elements, as we hypothesized, we analyzed our manipulation and then obtain its nucleotide sequence. Chem-seq results using peak-finding algorithms. We One of the first applications of NGS was ChIP-seq used HOMER [20] and MACS2 [21] for the sequence (chromatin immunoprecipitation followed by sequen- reads obtained from bead and quercetin–bead DNA li- cing). Recently, a new NGS application has been re- braries and selected significantly overlapping peak calls ported that allows extracting and sequencing DNA resulting from both algorithms. To exclude potential regions bound to small chemical molecules, namely bead signals from the quercetin treatment, we excluded Chem–seq (chemical affinity capture and massive paral- overlapping peak calls from beads and quercetin. Our lel DNA sequencing). In contrast to ChIP-seq, which final list contained 44,000 significant bead peaks and 1, targets DNA regions by using a specific antibody against 400 quercetin peaks. For downstream analysis, we chose a known chromatin, Chem-seq allows capturing chroma- the most significant 1,000 peaks from each list. We used tin regions bound to small molecules without prior in- GREAT [22] for enrichment analysis of quercetin and formation, using an unbiased, nonspecific marker such bead peaks up to 500 bp upstream of the transcription as biotin [17]. Biotin can be integrated into the molecule start site (TSS) and found that 36 genes from the quer- of interest, and then streptavidin can selectively filter cetin peak list corresponded to the cell cycle and cell only biotinylated molecules bound to chromatin regions division (Fig. 2c, Table 1). In contrast to the significant which can then be mapped to the genome. Although enrichment of genes found in the quercetin treatment, Chem-seq is a newly emerging NGS application, several the equivalent 1,000 bead peaks did not result in any reports have already illustrated its ability to isolate functional enrichment. To check for a potential DNA known drug–chromatin interactions [17, 18]. In the motif site for those 1,000 peaks, we used the HOMER present study, Chem-seq and RNA-seq were performed, de novo motif algorithm to scan for motifs in our data- and their data analyzed and combined to gain unique in- set and compare them with known HOMER motifs. Cor- sights into the interaction of quercetin molecules with responding with the gene-enrichment results, we the genome. We show that quercetin binds to mono- detected significant E2F DNA motif enrichment in the cytes’ chromatin and acts as a cis-regulatory element. In quercetin treatment (Fig. 2d). E2F is a gene family that particular, we show that quercetin modulates the expres- encodes transcription factors involved in cell-cycle regu- sion of genes involved in the cell cycle and cell develop- lation [23]. Importantly, no significant enrichment was ment. Our findings provide novel insights into the found in the beads-only treatment. mechanism by which the polyphenol quercetin affects monocyte biology. Quercetin alters cis-regulatory genes in monocytes To determine whether quercetin affects transcriptional reprogramming in cis, we performed RNA-seq for quer- Results cetin and dimethylsulfoxide (DMSO) treatments (see Biotinylated quercetin synthesis Methods), resulting in 16,308 coding genes, of which 1, Previous studies have shown that both the physico- 869 were found to have significant differential expression chemical properties and bioactivities of polyphenols with fold change (FC) between 0.5 and 2 and false dis- can be significantly improved via conjugation with covery rate (FDR) < 0.05. Quercetin is known to have a cleavable promoieties, such as pivaloxymethyl (POM) global effect on transcription, either directly or through or isopropyloxycarbonylmethoxy (POC) groups, to its modulators [10, 13]. The promotor enrichment results 7-OH position [19]. Based on these observations, we displayed ~ 36 cell-cycle genes with peaks

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