- Genome-wide localization of the. - Background: Quercetin is a polyphenol of great interest given its antioxidant activity and involvement in the immune response. - 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.. - Polyphenols in gen- eral, and quercetin in particular, readily penetrate cellu- lar and nuclear membranes and accumulate in the cell nucleus [5]. - In addition, polyphenols have been found to inter- act with various transcription factors and to regulate cell-cycle arrest, apoptosis and survival [10]. - In another study, we found that quercetin binds to an allosteric site of the serum en- zyme paraoxonase 1, affecting its function and biology [11]. - Experi- ments in the last 3 years have suggested that polyphenols bind to specific DNA-sequence motifs. - 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.. - 2 Faculty of Medicine in the Galilee, Bar-Ilan University, 1311502 Safed, Israel Full list of author information is available at the end of the article. - One of the first applications of NGS was ChIP-seq (chromatin immunoprecipitation followed by sequen- cing). - Biotin can be integrated into the molecule of interest, and then streptavidin can selectively filter only biotinylated molecules bound to chromatin regions which can then be mapped to the genome. - In the present study, Chem-seq and RNA-seq were performed, and their data analyzed and combined to gain unique in- sights into the interaction of quercetin molecules with the genome. - In particular, we show that quercetin modulates the expres- sion of genes involved in the cell cycle and cell develop- ment. - Biotinylated quercetin synthesis. - The biotinylated quercetin adduct was analyzed by high-performance liquid chromatography (HPLC) and liquid chromatog- raphy–mass spectrometry (LC–MS) (Additional file 1:. - Figure S1b), and found to correspond to the expected mass.. - Localization of quercetin genomic binding sites using Chem-seq. - We used GREAT [22] for enrichment analysis of quercetin and bead peaks up to 500 bp upstream of the transcription start site (TSS) and found that 36 genes from the quer- cetin peak list corresponded to the cell cycle and cell division (Fig. - In contrast to the significant enrichment of genes found in the quercetin treatment, the equivalent 1,000 bead peaks did not result in any functional enrichment. - Cor- responding with the gene-enrichment results, we detected significant E2F DNA motif enrichment in the quercetin treatment (Fig. - E2F is a gene family that encodes transcription factors involved in cell-cycle regu- lation [23]. - Importantly, no significant enrichment was found in the beads-only treatment.. - The promotor enrichment results displayed ~ 36 cell-cycle genes with peaks at ~ 500 bp from the TSS (Table 1). - levels of the background showed that quercetin tends to increase (P = 0.02) expression (median = 1.2) compared to the background (median = 1) (Fig. - To exclude randomal- ity, we used a permutation test of the FC values of the 36 empirical genes compared to a randomly selected group of genes (Fig. - In each cycle, we compared the empirical FC values to the FC of a random set of 36 genes using the Kolmo- gorov–Smirnov test and recorded the P values (denoted as Pempirical). - To exclude reproducibility of the test, we used the Kolmogorov – Smirnov test between two ran- dom selections of 36 genes and recorded the P values (denoted as Prandom). - This means that the FC values of the 36 genes are likely to be significantly different from those of a random set.. - These data sup- ported our hypothesis that cell-cycle gene expression is altered after quercetin induction.. - We compared the effect of biotinylated quercetin (Fig. - 2a) on gene expression with that of quercetin.. - We prepared another RNA-seq assay with monocyte cells, except that this time we used the biotinylated quercetin as the treatment and DMSO as the control and measured the FC values. - The FC values of the 36 genes under quercetin and biotinylated quercetin regulation were similar (P = 0.4, t test), but the latter were significantly different from the background gene expression (P <. - Our analysis thus showed that quercetin and biotinylated quercetin have similar effects on gene expression.. - Table 1 List of the 36 cell cycle genes and fold change (FC) after Quercetin treatment. - Polyphenols are the most abundant antioxidants in the human diet, found in fruit, vegetables, nuts and tea.. - Quercetin is one of the most abundant polyphenols and although its anti-inflammatory and anticancer properties have been well studied, much uncertainty surrounds its mechanism of action. - There is an overall tendency to ex- plain the beneficial effect of a polyphenol via its antioxi- dant activity, even if polyphenol concentrations in the blood barely reach the level needed for such activity (10–100 μM). - Even though the exact mechanisms by which quercetin exerts its beneficial effects remain un- clear, it is known to influence intracellular redox status, modulate gene expression of enzymes associated with biotransformation—such as phase I and phase II metab- olism, suppress cell proliferation by interacting with type II estrogen-binding sites, inhibit chromosomal alter- ations and tyrosine kinase, modulate several signal- transduction pathways through actions at MEK/ERK and Nrf2/keap1, and stabilize the tumor suppressor p53 at both the mRNA and protein levels to reactivate p53- dependent cell-cycle arrest and apoptosis. - The massive involvement of quercetin in cell activity requires deep investigation into its mechanism of action. - Monocytes were chosen be- cause of the ample literature on the effects of quercetin on signal transduction and gene expression (mostly anti- inflammatory and antibacterial genes) in monocytes and macrophages [25, 26]. - To examine direct binding sites of quercetin to the genome via the Chem-seq method, biotinylated quercetin was synthesized and evaluated (Fig. - Biotinylated querce- tin’s effect on cellular gene expression was examined in comparison to that of quercetin by comparing the expression levels of the 36 cell-cycle genes to the background gene expression after biotinylated quercetin induction (Table 1). - Thus, results from Chem-seq may reflect quercetin activity in the cell. - In particular, those binding sites were localized to the promotor regions of 36 genes that are mostly involved in cell-cycle pathways and skeletal and kidney development (Fig. - Moreover, the RNA-seq data illustrated that those 36 genes are significantly upregu- lated compared to the background genome (Fig. - 1 Synthesis of biotinylated quercetin. - The biotinylated quercetin was synthesized in 5 steps (see Methods), and compounds are labeled from 1 to 6. - 2 Colocalization of quercetin beads with DNA and the 1,000 most significant peaks across the genome. - a Structure of biotinylated quercetin product. - b Flowchart of Chem-seq procedure. - c Gene ontology (GO) enrichment of the 36 genes associated to the quercetin peaks up to 500 bp from the transcription start site. - P empirical denotes the distribution of P values obtained by comparing the empirical FC values to the random set. - b Kernel density estimate of log FC of quercetin and the background set. - Quercetin denotes the FC values of 36 cell-cycle genes. - Our list of candidate genes was consistent with the literature showing that quercetin exerts a direct, proapoptotic effect in tumor cells and may block the growth of several human cancer cell lines at different phases of the cell cycle. - We also found that such regions are enriched with DNA motifs of the transcription factor family E2F (Fig. - 2d) with particular similarity to the transcription factor E2F7 which is known to be involved in the S-phase of cell- cycle regulation [28]. - This result is supported by the lit- erature as quercetin is well documented to regulate, dir- ectly or indirectly, cell-cycle and anticancer genes. - Some of the 36 genes that are postulated to be under cis-regulation of quercetin have been previ- ously reported. - For example, the FOXO transcription factor family [30]: 2 genes on our candidate gene list be- long to the FOXO transcription factors (FOXD1 and FOXJ1. - 2) and may be under direct regulation of quercetin. - Moreover, our list includes 2 genes belonging to the mitogen-activated protein ki- nases, MAPK14 and MAPK9, FC >. - Those genes are well known for their involvement in resistance to colon cancer, cell-cycle arrest and cell death [35, 36].. - Although the precise mechanism of quercetin activity has yet to be understood, there is vast evidence in the scientific literature of this polyphenol regulating various gene families. - Further genome-level investiga- tion and mapping of the direct interactions of polyphenols and chromatin genome-wide could provide new insights into the mechanisms by which a small. - Biotinylated quercetin synthesis (Fig. - Biotinylated quercetin adduct (6). - With- out further purification, acetonitrile (3 mL) and 3 M aque- ous HCl (3 mL) were added to the reaction mixture and the resulting solution was stirred and refluxed for 1 h.. - yield) as a pure bright-yellow solid (one peak appeared in the HPLC chromatogram of the product, Additional file 1:. - This product was identified as biotinylated quercetin adduct (6) by LC–MS analysis (Additional file 1:. - Chem-seq. - Cell nuclei were prepared using the EZ-Magna Chip Kit (Mercury) as proposed in the kit protocol. - Then, magnetic strepta- vidin dynabeads (MyOne Streptavidin T1, Invitrogen) were preincubated in PBS containing 0.5% bovine serum albumin and either 200 μM biotinylated quercetin or ve- hicle (DMSO) for 6 h. - Contaminating RNA and protein were digested by addition of RNase and proteinase K, respectively, and the DNA was purified as described in the kit. - Size and quality of the samples were examined with a bioanalyzer (Agilent Technologies).. - RNA-seq. - Chem-seq mapping and peak-calling procedure. - Chem-seq data of quercetin, bead treatments and input DNA were aligned to the human reference genome hg19 using Bowtie2 [38]. - To explore potential binding sites of quercetin to the DNA, we used the peak-finding algorithms MACS2 [21] and the Hypergeometric Optimization of Motif Enrichment (HOMER) peak-find- ing program [20].. - (i) biotinylated quercetin–streptavidin beads, (ii) strepta- vidin beads, (iii) input DNA. - The peak-calling procedure of MACS2 and HOMER was performed as follows: first, DNA alignment of the biotinylated quercetin as the treatment and streptavidin beads as the control was used to prepare a list of differential biotinylated quercetin peaks. - As part of this procedure, the biotinylated quercetin bead and the streptavidin bead DNA read alignments were used as the treatment sam- ple, while the input reads were used as the control. - RNA-seq data were aligned to the human reference gen- ome hg19 using Bowtie2 [38]. - Biotinylated quercetin analysis. - a HPLC chromatogram of the biotinylated quercetin adduct (C 29 H 33 N 3 O 9 S, Mw g/mol) using an RP-18 column. - b LC – MS analysis of the product using Q-TOF LC – MS with positive electrospray ionization (ESI+) method. - Two peaks appeared in the m/z spectra of the product, at 600.2033 (M + H. - SK performed the Quercetin-Biotin synthesis and verified by analytical methods and provided critical feedback in the project. - 2 Faculty of Medicine in the Galilee, Bar-Ilan University, 1311502 Safed, Israel. - Effect of quercetin on experimental hyperlipidemia and atherosclerosis in rabbits. - Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells. - Quercetin, a natural flavonoid interacts with DNA, arrests cell cycle and causes tumor regression by activating mitochondrial pathway of apoptosis.. - E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. - E2F7 represses a network of oscillating cell cycle genes to control S-phase progression. - Quercetin induces apoptosis and cell cycle arrest in triple-negative breast cancer cells through modulation of Foxo3a activity. - The role of the collagen matrix in skeletal fragility. - Regulation of the hepatocyte cell cycle by type I collagen matrix: role of cyclin D1
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