Sevki Arslan

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

A Comparative Study for the Evaluation of Two Doses of Ellagic Acid on Hepatic Drug Metabolizing and Antioxidant Enzymes in the Rat

The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.

Alteration of drug metabolizing enzymes in sulphite oxidase deficiency

The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments.

Association of the CYP1A1*2A, GSTT1 null, GSTM1 null, mEPHX*3, and XRCC1-399 genetic polymorphisms with ulcerative colitis.

Dear Editor: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that affects the large intestine and rectum. IBDs are believed to result from the interplay between a number of disease genes and environmental factors. Epidemiological data provide evidence that genetic predisposition to ulcerative colitis depends on the contribution of multiple genes rather than a single genetic factor. Recently, the number of ulcerative colitis-associated loci and genes, such as ECM1, IL23R, IL12B, HLA, NKX2-3, and VDR, has increased. However, mutations within these genes were absent in the majority of patients, suggesting the presence of additional susceptibility genes. It is suggested that reactive molecules, such as reactive oxygen species ROS, may play a pivotal role in the mechanism of inflammation by altering the intestinal permeability, which leads to tissue injury in the mucosa, ultimately resulting in tissue damage. Reactive molecules may be products of the endogenous metabolism of xenobiotics catalyzed by biotransformation enzymes. Changes in detoxification of xenobiotics that cause epithelial damage may confer susceptibility to UC. Chronic inflammation of the colon may further lead to carcinogenesis and tumor formation. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of UC and colon cancer. Biotransformation enzymes such as cytochrome P450s and GSTs perform vital roles in the metabolism of xenobiotics and ROS. They often convert parent compounds into highly reactive metabolites, such as epoxides, that may bind to cellular components and induce tissue damage and carcinogenesis. Detoxification of these reactive metabolites is carried out by either conjugating with glutathione, which is catalyzed by GSTs, or by hydration, which is catalyzed by epoxide hydrolases. X-ray repair cross-complementing groups (XRCCs) are important proteins of the DNA repair pathways. The XRCC1 gene is responsible for a scaffolding protein that directly associates with the processes of base excision repair or single-strand break repair. Numerous polymorphisms that result in altered enzyme activities have been described in the genes of these enzymes. It has been suggested that individual variations in the susceptibility to the mutagenic and carcinogenic activity of xenobiotics may be partially explained by differences in their activation and deactivation pathways. As chemical and oxidative stress may be involved in the etiology of ulcerative colitis, polymorphic genes encoding for these biotransformation enzymes may modulate the genetic susceptibility to ulcerative colitis and carcinogenesis in the colon. Therefore, this study was undertaken to determine whether genetic polymorphisms in biotransformation enzymes in patients with ulcerative colitis differ from those in healthy controls. Polymorphisms in genes encoding cytochrome P450 1A1 (CYP1A1), glutathione S-transferase mu-1 (GSTM1) and theta-1 (GSTT1), microsomal epoxide hydrolase (EPXH) and X-ray repair complementing group 1 (XRCC1) were investigated. The present case–control study was performed in subjects recruited between 2008 and 2010. A total of 161 consecutive patients with ulcerative colitis (94 males, 67 females; all Caucasian) consulting the outpatient clinic of the Department O. Buyukgoze : S. Arslan :A. Sen (*) Biology Department, Faculty of Arts & Sciences, Pamukkale University, Kinikli Campus, Kinikli, 20070, Denizli, Turkey e-mail: sena@pau.edu.tr

Modulatory role of GSTM1 null genotype on the frequency of micronuclei in pesticide-exposed agricultural workers

In this study, we aimed to investigate the extent of genotoxic risk and the association between null GSTM1/GSTT1 and GSTP1 Ile105Val variants and cellular DNA damage, as measured by micronucleus (MN) assay in a group of agricultural workers from Denizli, Turkey. Peripheral blood samples were collected from 116 subjects, including 58 workers who were occupationally exposed to pesticides and 58 healthy unexposed controls. The MN frequencies of each individual were assessed by cytokinesis-blocked micronuclei assays on lymphocytes. Genotypes for different GST variants were determined using polymerase chain reaction-based methods. A significant 3.4-fold increase in MN frequency was observed in workers compared with the controls (p < 0.001). Among the GST genotypes, only the GSTM1 null genotype was found to be significantly associated with an increased MN frequency in workers (p = 0.01). Individuals with a concomitant null GSTM1/GSTT1 genotype demonstrated a significant (p = 0.01) increase in MN frequency compared with those with functional isozymes in the exposed worker group. The association of the GSTM1 null genotype with higher MN frequency suggests that it may be a modifier of genotoxic risk in individuals exposed to pesticides and may thus be a candidate susceptibility biomarker for human biomonitoring studies.

Temporal and Spatial Epigenome Editing Allows Precise Gene Regulation in Mammalian Cells

Cell-type specific gene expression programs are tightly linked to epigenetic modifications on DNA and histone proteins. Here, we used a novel CRISPR-based epigenome editing approach to control gene expression spatially and temporally. We show that targeting dCas9-p300 complex to distal non-regulatory genomic regions reprograms the chromatin state of these regions into enhancer-like elements. Notably, through controlling the spatial distance of these induced enhancers (i-Enhancer) to the promoter, the gene expression amplitude can be tightly regulated. To better control the temporal persistence of induced gene expression, we integrated the auxin-inducible degron technology with CRISPR tools. This approach allows rapid depletion of the dCas9-fused epigenome modifier complex from the target site and enables temporal control over gene expression regulation. Using this tool, we investigated the temporal persistence of a locally edited epigenetic mark and its functional consequences. The tools and approaches presented here will allow novel insights into the mechanism of epigenetic memory and gene regulation from distal regulatory sites.

Capparis ovata treatment suppresses inflammatory cytokine expression and ameliorates experimental allergic encephalomyelitis model of multiple sclerosis in C57BL/6 mice.

Since ancient times, Capparis species have been widely used in traditional medicine to treat various diseases. Our recent investigations have suggested Capparis ovatas potential anti-neuroinflammatory application for the treatment of multiple sclerosis (MS). The present study was designed to precisely determine the underlying mechanism of its anti-neuroinflammatory effect in a mouse model of MS. C. ovata water extract (COWE) was prepared using the plants fruit, buds, and flower parts (Turkish Patent Institute, PT 2012/04,093). We immunized female C57BL/6J mice with MOG35-55/CFA. COWE was administered at a daily dose of 500mg/kg by oral gavage either from the day of immunization (T1) or at disease onset (T2) for 21days. Gene expression analysis was performed using a Mouse Multiple Sclerosis RT² Profiler PCR Array, and further determinations and validations of the identified genes were performed using qPCR. Whole-genome transcriptome profiling was analyzed using Agilent SurePrint G3 Mouse GE 8X60K microarrays. Immunohistochemical staining was applied to brain sections of the control and treated mice to examine the degree of degeneration. COWE was further fractionated and analyzed phytochemically using the Zivak Tandem Gold Triple Quadrupole LC/MS-MS system. COWE remarkably suppressed the development of EAE in T1, and the disease activity was completely inhibited. In the T2 group, the maximal score was significantly reduced compared with that of the parallel EAE group. The COWE suppression of EAE was associated with a significantly decreased expression of genes that are important in inflammatory signaling, such as TNFα, IL6, NF-κB, CCL5, CXCL9, and CXCK10. On the other hand, the expression of genes involved in myelination/remyelination was significantly increased. Immunohistochemical analysis further supported these effects, showing that the number of infiltrating immune cells was decreased in the brains of COWE-treated animals. In addition, differential expression profiling of the transcriptome revealed that COWE treatment caused the down regulation of a group of genes involved in the immune response, inflammatory response, antigen processing and presentation, B-cell-mediated immunity and innate immune response. Collectively, these results suggest anti-neuroinflammatory mechanisms by which COWE treatment delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

Anti-neuroinflammatory effect of butanolic subextract of Capparis ovata water extract used as an alternative and complementary treatment for multiple sclerosis

compared with that of control siRNA. Collagen I, III, IV, V and decorin expression were found in the perivascular space and in the parenchyma. Collagen I, IV and V are expressed by astrocytes which were also expressing RGC-32. Since RGC-32was found to be involved inmediation of TGF-beta effects, we investigated its role in TGF-beta-induced ECM expression and reactive astrocyte marker α-smooth muscle actin (αSMA). In cultured astrocytes, α-SMA, collagen I, IV and V as well as fibronectin were significantly induced at 18 h of stimulation with TGFbeta. Next, we silenced RGC-32 expression by transfecting astrocytes with siRGC-32 and compared the effect of this treatment to that of control siRNA. We found that α-SMA expression was significantly reduced after RGC-32 silencing (p b 0.05). In addition we found that RGC-32 silencing resulted in a significant reduction in TGF-beta-induced collagen I (p b 0.01), collagen IV (p b 0.02), collagen V (p b 0.05) and fibronectin (p b 0.05) expression. Using astrocytes isolated from RGC-32 knockout (KO) mouse we found that TGF-beta-induced collagen IV and alpha-SMA expression were significantly reduced in RGC-32 KO when compared with wild type mouse. The effect of RGC-32 silencing on alpha-SMAexpression suggests that RGC-32 is required for the transition of astrocytes to a reactive state. Our data also indicate that RGC-32 plays an important role in the TGF-beta-mediated induction of ECMexpression in astrocytes. RGC-32 may therefore represent a useful new target for therapeutic intervention in MS.