Epigenetic therapy of novel tumour suppressor ZAR1 and its cancer biomarker function
Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation.
We are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines.
ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.
KeywordsCancer biomarker ZAR1 Tumour suppressor DNA methylation Epigenetics p53 Zinc finger Epigenetic editing CRISPR-Cas9
Clustered regularly interspaced short palindromic repeats
Zygote arrest 1
Cancer remains a devastating disease with 17 million new cases and 9.6 million deaths each year worldwide, as well as an expected continued rise of cases . The total economic cost of cancer was estimated to be US$ 1.16 trillion , and only 1.4% of this staggering number is spent on cancer research . Lung cancer remains the leading cause of cancer death by far , and we published that ZAR1 is a novel tumour suppressor in lung cancer . Zygote arrest 1 (ZAR1) was initially reported to be a maternal-effect gene critical for oocyte to embryo transition in mouse ; however, we and others reported that its expression is not only limited to the oocyte but also found further tissues. ZAR1 was reported to be expressed in porcine and bovine brain and testis , bovine heart and muscle , human lung , and rabbit lung . Human ZAR1 locates on chromosome 4 (4p11) and harbours a large 1.5 kb CpG island (CGI; Additional file 1: Figure S1a). CpG islands are genomic regions defined by the enrichment of CpG dinucleotides . ZAR1 codes for a 1275 nt transcript (4 exons) and a 424 aa protein with a carboxy (C)-terminal zinc-finger (CpG plot, NCBI, and UCSC genome browser; Additional file 1: Figure S1a). In the context of cancer, evidence has been growing for a role of ZAR1, even though early reports were in part contradicting. In melanoma, the methylation of exon 1 was reported, but ZAR1 was said to be overexpressed in some hypermethylated melanoma cell lines . In brain tumours and neuroblastoma, non-promoter methylation was reported [12, 13], as was its absence in hypermethylated glioma cell lines . In hypermethylated neuroblastoma, however, expression of ZAR1 was detected and indicated that ZAR1 knockdown promotes differentiation in neuroblastoma cells . Intragenic ZAR1 methylation decreased in high-grade vs. low-grade tumours of the bladder . In hepatitis C virus, positive liver cancer ZAR1 was reported to be methylated in exon 1 . In cervical cancer, ZAR1 was methylated vs. normal epithelia . With the present work, we report ZAR1 as a cancer biomarker and also elucidate its role in human cancer using state-of-the-art methylation sequencing, transcriptomic approaches, mass spectrometry for the identification of interacting partners, and epigenetic reactivation by CRISPR-dCas9.
ZAR1 is a lung and kidney cancer biomarker
Our focus is the exciting and novel role of ZAR1 as a tumour suppressor in humans. ZAR1 is differentially expressed across human tissues (testis, colon, kidney, lung, skin, and brain; Additional file 1: Figure S1b) and not restricted to the ovary (set 1 for comparability). Exploring a possible ZAR1 function in cancer, we found that ZAR1 expression (n = 917, CCLE Cancer Cell Line Encyclopedia) significantly correlated with genes that carry the GO-terms ‘regulation of RNA metabolic processes’, ‘cell communication’, ‘signal transduction’, ‘cell–cell signalling’, ‘anatomical structure development’, and ‘embryonic morphogenesis’ (Additional file 1: Figure S1c). We earlier reported that ZAR1 is epigenetically regulated in lung cancer . Here, we add evidence for ZAR1 hypermethylation in further cancer types (Additional file 1: Figure S2a, b). ZAR1 is methylated in various cell lines, in all germ cell lines (n = 14), in half of the malignant melanoma (n = 4) and kidney cancer cell lines (n = 4), in all mamma (n = 3), and 90% of brain cancer cell lines (glioblastoma; n = 8). In ovarian carcinoma, ZAR1 hypermethylation increases from 15% in primary tumours (n = 20) to 67% in cancer cell lines (n = 6), whereas the controls were unmethylated (Additional file 1: Figure S3a, b). Quantification of ZAR1 methylation revealed a methylation threshold at 20% (Additional file 1: Figure S3c).
ZAR1 is highly conserved and an mRNA binding protein
ZAR1 tumour suppressor function is zinc-finger and p53 dependent
ZAR1 is associated with ribosomal/mRNA networks, zinc-finger dependent
Epigenetic therapy reactivates the ZAR1 tumour suppressor
In summary, we report here that ZAR1 is epigenetically inactivated across cancers and has prognostic value for lung and kidney cancer as a biomarker. We show evidence for its tumour suppressor function, depending on its zinc-finger domain and its mRNA binding ability. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of the ZAR1 tumour suppressor is feasible.
ZAR1 came to our attention due to a 450k methylation array in which we identified it as one of the most strongly methylated target genes in a lung cancer cell line . Since then we were curious to understand its epigenetic inactivation in cancer and its role in carcinogenesis. ZAR1 is not only strongly hypermethylated across various cancers types but also across its complete CGI. We also show that ZAR1 methylation is a suitable biomarker for lung and kidney cancer. Our results clarify the in part contradicting earlier reports on ZAR1 methylation. In cancer, ZAR1 is under an epigenetic control, which is a common theme for tumour suppressors in carcinogenesis . We did not observe frequent mutation events in ZAR1 and conclude that hypermethylation is the dominant inactivating mechanism in cancer. Using epigenetic editing, we were able to modulate ZAR1 expression and methylation in cancer cell lines, further proving its epigenetic inactivation mechanism. Using TET1 as an epigenetic modifier, we could even show the demethylation of the ZAR1 promoter that reactivated ZAR1 expression. The use of epigenetic therapy in the reactivation of tumour suppressors has been discussed recently [40, 41]. We believe that our findings are suggesting ZAR1 reactivation in cancer as a promising target to such intervention.
With the present study, we demonstrated tumour suppressor properties of ZAR1, which were dependent on its zinc-finger domain. We also showed that ZAR1 is an mRNA binding protein and ZAR1 associates with the mRNA/ribosomal/translational network, depending on its zinc-finger. We hypothesise that ZAR1 binds its mRNA targets and thereby regulates their translation. In the future, we intend to explore the role of ZAR1's zinc-finger-dependent dimerization and post-translational regulation in its mRNA binding ability, as well as the mRNA-binding-dependent interactome. Our kinase predictions based on sequence references are hinting towards phosphorylation of ZAR1, which may regulate its stability or interaction with partners. Further studies will reveal if and how ZAR1 is controlled post translation. We believe that our work proves that, beyond its initially reported growth-controlling role in the oocyte , ZAR1 has an exciting additional role in tissues, where it controls cellular growth and contributes to cancer suppression. ZAR1 promoter hypermethylation and subsequent epigenetic inactivation of ZAR1 on the other hand contributes to carcinogenesis.
We have discovered ZAR1 as a potential cancer biomarker, which should be followed by assay development and analytical validation, clinical utility validation, and ultimately clinical implementation . We suggest that determining the ZAR1 methylation levels could serve as a convenient biomarker in the future. The advantages of DNA as a biomarker is its superior stability in cells and in body fluids, where free circulating DNA is present . Methylation of DNA is a covalent bond, stable and well detectable by the bisulfite conversion method . Bisulfite treatment of DNA, the gold standard for DNA methylation analysis [44, 45, 46], high-throughput bisulfite conversion  as well as digital droplet PCR (amplification of low levels of DNA in disproportionate sample/target combinations)  are available. DNA samples may be taken from tumour resections, biopsies, or from liquid biopsy material as a non-invasive method . Circulating tumour cells or circulating tumour DNA are present in blood, body fluids, or even in exhaled breath condensates . The latter are in clinical trials [51, 52], and liquid biopsies are FDA approved (lung cancer EGFR mutation tests as companion diagnostic) . We believe that in the future, also ZAR1 methylation has the potential to be part of cancer screens. The FDA has already approved of several cancer biomarkers that are in clinical practice for, e.g., liver, prostate, ovarian, breast, pancreatic, lung, and thyroid cancer [54, 55], and there is a DNA methylation marker screening available for colorectal cancer, which is blood based . In our study, we show that epigenetic therapy of ZAR1 is achievable. In the future, we are anticipating that targeted therapies will also include epigenetically inactivated tumour suppressors by, e.g., the CRISPR-dCas9 technique and viral application of epigenetic editors to reactivate not only ZAR1 in vivo in cancer.
For the first time, our study presents evidence that ZAR1, which harbours tumour suppressive properties, is a prognostic and diagnostic cancer biomarker. ZAR1 suppresses tumour cell line growth in part through p53 and strongly depending on its functional zinc-finger. Ultimately, we found that ZAR1 can be reactivated by epigenetic therapy using the CRISPR dCas9 system.
Methylation analysis. CpG Island prediction, PCR product size, and digestion products
The promoter region of ZAR1 was analyzed by CpG plot http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/ and UCSC genome browser. Primers for bisulfite-treated DNA were designed to bind only fully converted DNA and amplify promoter region. The precise promoter region was chosen for CpG content and presence of according restriction enzymes for CoBRA analysis. The size of the ZAR1 CoBRA PCR product is 186 bp (with TaqI site at 89). For further details on CoBRA analysis see Richter et al.'s study .
DNA Isolation, CoBRA, and Pyrosequencing
DNA was isolated after proteinase K (Thermo Fisher Scientific) digest and extracted either with phenol/chloroform or by QIAamp DNA extraction kit (Qiagen), and concentrations were determined. For CoBRA methylation analysis, a total of 2 μg genomic DNA was bisulfite treated (5 mM hydroquinone, 1.65 M sodium metabisulfite, and pH 5.5 with 0.025 M NaOH) and incubated overnight at 50 °C. DNA was purified using MSB Spin PCRapace (STRATEC Molecular), eluted in 50 μl H2O, and followed by 10 min incubation with 5 μl 3 M NaOH at 37 °C. DNA was then precipitated with 100% ethanol and ammonium acetate and resolved in 1 × TE buffer. Alternatively, we used 500 ng genomic/plasmid DNA and the EZ DNA methylation kit (Zymo research) according to manufacturer's protocol. Bisulfite DNA was used for CoBRA PCR. The subsequent PCR product (CoBRA primers) was digested with 0.5 μl of TaqI (Thermo Fisher Scientific) 1 h at 65 °C and resolved on 2% TBE gel (×0.5) together with mock control and DNA ladder. Pyrosequencing (incl. five CpGs) was performed according to manufacturer's protocol with PyroMark Q24 System (Qiagen). When analysing methylation of the artificial ZAR1 promoter by pyrosequencing the 2, CpG was not present due to mutation of the cloned ZAR1 promoter. This allowed us to distinguish between genomic and ZAR1pRLnull plasmid being isolated and pyrosequenced. In vitro, methylation (pos. control) of genomic DNA was performed using CpG Methyltransferase M.SssI (NEB) according to manufacturer's protocol.
RNA expression analysis
RNA was isolated from human cell culture using Isol-RNA lysis procedure (Trizol, Thermo Fisher Scientific). RNA was DNaseI (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega). Quantitative RT–PCR was performed in triplicate with SYBR select (Thermo Fisher Scientific) using Rotor-Gene 3000 (Qiagen) and normalised to GAPDH/ACTB. We performed RNA microarrays (Clariom S human) according to manufacturer's protocol (P/N 703174 Rev. 2) with 200 ng of total RNA. Reagents/equipment were GeneChip WT PLUS Reagent Kit, P/N: 902280; GeneChip Hybridization, Wash, and Stain Kit P/N 900720, GeneChip Scanner, GeneChip Fluidics Station 450, GeneChip Hybridization Oven 640, Bioanalyzer 2100 (Agilent), and RNA600 NanoKit (Agilent).
Identification of RNA binding using crosslinking and immunoprecipitation (CLIP)
Potential binding of Zar1 to RNA in general and specifically to the WEE1 mRNA was tested using a shortened version of the CLIP procedure . HEK293T cells were transfected with ZAR1-EYFP or EYFP-empty for 30 h and UV irradiated (254 nm; 300 mJ/cm2) to crosslink RNA binding proteins to cellular RNA. After cell lysis, the RNA is trimmed by limited RNaseI digestion. ZAR1 is immunoprecipitated by GFP Trap (ChromoTek). The 2′,3′-cyclic phosphate produced by RNaseI digestion is removed by phosphatase treatment. The RNA is radioactively 5′ end-labelled with 32P. Free RNA is removed by gel electrophoresis followed by transfer to a nitrocellulose membrane, which binds proteins unspecifically. After autoradiography, the area with the covalent protein/RNA complexes of interest is cut from the membrane. The RNA is eluted from the membrane by protein digestion with proteinase K. RNA is reversely transcribed before RT-PCR.
Cell lines, lung cancer tissues, and controls
Cell lines used were published earlier. FTC-133 , Hep2 , Hep2G , A549 , HCT116wt, and HCT116delp53 were obtained from Thorsten Stiewe . RD [64, 65], germ cell carcinoma , malignant melanoma cell lines , kidney cancer cell lines , breast cancer cell lines , glioblastoma cell lines were obtained from Lienhard Schmitz , ovarian carcinoma samples, and control patient material; and all patients signed informed consent before enrolment . The study was approved by local ethic committees .
Cell culture, cell cycle analysis, and ZAR1 localisation
Cell lines were grown in appropriate medium (DMEM or RPMI) supplemented with 10% FCS and 1% Penicillin/Streptomycin under cell culture conditions (37 °C, 5% CO2). Cell lines were transfected using Turbofect (Thermo Fisher Scientific), X-tremeGENE HP (Roche), or Polyethylenimin (Sigma) with either 4 μg (6 wells) or 10 μg (10-cm dishes). Regarding flow cytometry analysis, cells were transfected and ethanol fixed at indicated time points. The following day, cells were treated with 50 μg/ml RNaseA for 30 min at 37 °C. Subsequently, cells were stained with 50 μg/ml propidium iodide prior to measuring DNA content in FACSCantoII (BD Biosciences). FACSDiva Software (BD Biosciences) was used for measurement/gating to distinguish transfected fluorescent cells and to determine cells in G0/G1, S, and G2/M phases of the cell cycle. For localisation analysis, cells were seeded on glass slides and transfected the following day. Cells were fixed with 3.7% formaldehyde at according time points, permeabilised using tritonX, stained with DAPI (0.1 μg/ml in PBS, Sigma), embedded in anti-fading with Mowiol (Sigma), and analysed with Axio Observer Z1 (Zeiss) under ×63 magnification and Volocity Software (Perkin Elmer). Analysis of ZAR1 was restricted to overexpressed ZAR1 due to commercial antibodies not being useful for endogenous ZAR1 detection in western blotting and immunofluorescence.
Plasmids and promoter reporter assay
ZAR1 coding sequence was cloned into pEGFP-C2  (Clontech), pEYFP (Clontech), pCMVTag1 (Flag; Agilent), and pEBG (GST+Flag). The ZAR1 zinc-finger was deleted by site-directed mutagenesis (QuikChange Lightning, Agilent) and verified by sequencing, western blotting, and fluorescence microscopy. The ZAR1 promoter (position −530 to +76 relative to transcriptional start site) was amplified from genomic DNA and cloned into pRL-null (Promega) . We used the Dual-Glo-Luciferase Reporter Assay System (Promega) according to manufacturer's protocol and pGL3 for transfection/expression control. Cell lines were chosen due to determined ZAR1 status (methylation and expression) and being well established cancer cell line models. Cell lines show a superior transfection efficiency. HEK, HeLa, or HCT116 cells were transfected for 24 h with pRLnull empty or ZAR1 promoter containing pRLnull together with ZAR1 guides in px549dCas or empty-px549dCas, and together with epigenetic effectors or empty-pcDNA3.1 control and with pGL3 transfection control. Lysates were prepared after 24 h, and luciferase was measured. Renilla luciferase promoter results (pRLnull) were normalised to pGL3 (firefly luciferase). ZAR1pRLnull was normalised to pRLnull empty. Measurements were done in triplicates. Controls were set 1. Transfections were performed at 80% cell density in 6 wells with a total of 4μg Plasmid using PEI.
Epigenetic editing/ Epigenetic therapy by CRISPR-dCas9
CRISPR-Cas9 vector px549 was obtained from Lienhard Schmitz (Giessen, Germany) and adapted for epigenetic editing by inactivation of Cas9 (dCas9 site-directed mutation). ZAR1 guide RNAs/Oligos were positioned/generated using Benchling (Additional file 1: Figure S1a)  and cloned into px549-dCas and TET1 through the BbsI site. Epigenetic modifier plasmids were ordered from Addgene and modified if indicated: pcDNA-dCas9-p300 Core (61357), pdCas9-DNMT3A-EGFP (71666) with deletion of U6 promoter (site-directed mutagenesis), pdCas9-Tet1-CD (83340) as wildtype, with deletion of U6 promoter (site-directed mutagenesis) or as wildtype with cloned ZAR1 guides in BbsI restriction site (ZAR1-guided-TET1), pcDNA3.1-MS2-Tet1-CD (83341), Ezh2[SET]-dCas9 (100087), DNMT3A-dCas9 (100090). Epigenetic editing of endogenous ZAR1 was performed in the ZAR1 partially methylated Hela, if not mentioned otherwise. ZAR1 RNA guides are #1 ACTTTCGCTCACTTAGCCAG, #2 TGGTTCCCTTACGGATCAGC, #3 GTAGGGAGAAGGACGAAGAG, #4 GTCGCCTATTTAGGGTGCGG, #5 CGCGGCCACCAAGGGCAAGG, and #6 CCGCGGTACAGTGCTCGCTG and are positioned relative to TSS at −402 #1, −230 #2, −133 #3, −3 #4, +120 #5, and +386 #6.
Binding partner identification using GFP Trap and mass spectrometry
ZAR1-EYFP, ZAR1delZF-EYFP vs. EYFP-empty were overexpressed in HEK293T cells (24 h), and pulldown was performed according to manufacturer's protocol by GFP Trap (ChromoTek). Triplicate sample pairs were processed by off-bead digest, strong anion exchange (SAX) extraction, and dimethyl-labelling, followed by LC-MS2. I brief, beads were resuspended in two volumes urea buffer (6 M urea, 2 M thiourea, 10 mM dithiothreitol, 10 mM HEPES, pH 8.0) and incubated shaking for 30 min at room temperature. Cysteins were alcylated at 55 mM final concentration of iodoacetamide, shaking at room temperature and in the dark for another 30 min. Peptidolysis was then initiated with 0.5 μg Lys-C (Wako Chemicals GmbH) for 3 h shaking at room temperature, followed by dilution to 2 M urea/thiourea, addition of 0.5 μg trypsin (Serva) and an overnight shaking incubation at room temperature. Peptide-containing supernatants were brought to 1% NH3 and loaded onto three-layer SAX tips equilibrated previously with 30 μl of 0.1% NH3. After sequential washes with 30 μl 0.1% NH3 and 30 μl NH3 in 2-propanol, respectively, columns were syringe dried, peptides eluted using 30 μl 80% acetonitrile, 0.1% formic acid and vacuum dried. In-solution chemical labelling was performed as described [71, 72]. Peptides were resolubilized and acidified using a final concentration of 0.1% TFA. Free amines were differentially modified by reductive dimethylation. The labelling reaction was quenched on ice using ammonia solution and formic acid. Differentially labelled samples were mixed 1:1 by volumes and desalted on oligo R3 columns. The subsequent LC-MS2 analysis used an in-house packed 70 μm ID, 15 cm reverse phase column emitter (ReproSil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch GmbH) with a buffer system comprising solvent A (5% acetonitrile, 1% formic acid) and solvent B (80% acetonitrile, 1% formic acid). Relevant instrumentation parameters are extracted using MARMoSET  and included in the supplementary material. Peptide/protein group identification & quantitation was performed using the MaxQuant suite of algorithms [74, 75] (v. 18.104.22.168) against the human uniprot database (canonical and isoforms; downloaded on 2019/01/23; 169,389 entries) using the parameters documented in the supplementary material.
Further analysis of publicly accessed data and origin of data
Gene expression, promoter methylation correlation, and Kaplan–Meier calculations were performed using R2 Genomics Analysis and Visualization Platform , Wanderer , KM Plotter [78, 79, 80, 81], and MethSurv . Gene set enrichment analysis was performed using GSEA . The following are listed in order of appearance with resource of data. Additional file 1: Figure S1 ZAR1 expression in human normal tissues, HPA RNA-seq normal data, Bioproject PRJEB4337, data . ZAR1 expression correlation in Cellline CCLE Cancer Cell Line Encyclopedia - Broad - 917 - MAS5.0 - u133p2, log2, ZAR1 (1555775_a_at) APS = 16.2 (407) Avg = 12.8, Source: GEO ID: gse36133 Dataset Date: 2012-03-20. Additional file 1: Figure S4 ZAR1 expression in cancer cell lines vs. normal tissues, 1555775_a_at, log2, data Roth vs. Broad, Anova one way. ZAR1 methylation in normal to tumour tissues and cancer cell lines, cg22773661/cg1753764, data Lokk vs. Heyn vs. Esteller, Anova one way. ZAR1 methylation in normal to tumour tissues and cancer cell lines relative to CpG island/shores and for all ZAR1 (cg) reporters from array. T-SNE analysis on Broad and Roth, Transform: zscore, no gene filter, no sample filter, perplexity = 50, Colour mode: Colour by Gene (ZAR1), Transform log 2. Overview on R2 used datasets (class,tissue,disease+additional info-author-#samples-normalisation-platform): Normal Various - Roth - 504 – MAS5.0 - u133p2, Source: GEO ID: gse7307 Dataset Date: 2007-04-09; Cellline CCLE Cancer Cell Line Encyclopedia - Broad - 917 - MAS5.0 - u133p2, Source: GEO ID: gse36133 Dataset Date: 2012-03-20; Normal Tissues - Lokk - 70 - custom - ilmnhm450, Source: GEO ID: gse50192 Dataset Date: 2014-02-26; Tumor Types (landscape) - Heyn - 493 - custom - ilmnhm450, Source: GEO ID: gse76269 Dataset Date: 2017-06-07; Cellline Cancer Pharmacogenomic - Esteller - 1028 - custom - ilmnhm450, Source: GEO ID: gse68379 Dataset Date: 2016-07-05. Figure 1: Analysis performed using Wanderer  TCGA data, gene: ZAR1, dataset project: TCGA, data type: 450k Methylation Array, for LUAD lung adenocarcinoma and KIRC Kidney renal clear cell carcinoma. Pan-cancer mRNA RNA-seq using KM Plotter , Tumor type: Kidney renal clear cell carcinoma and Lung adenocarcinoma, Split patients by: Auto select best cutoff, Follow up threshold: 60 months. Analysis performed using MethSurv  on TCGA cancer datasets: KIRC Kidney renal clear cell carcinoma and LUAD Lung adenocarcinoma, Relation to island: Island, Genomic Region: TSS200, Split by: mean. Additional file 1: Figure S5: Conservation and alignment of by PhyloP; UCSC genome browser  and BioEdit  matrix: BLOSUM62 on sequence ZAR1 from homo sapiens, mus musculus and xenopus laevis from NCBI RefSeq . Swiss-Model  prediction by template ‘Pre-mRNA-splicing factor SLT11’ with sequence identity 22.22% in the range 318-380 aa and sequence similarity 0.33. PTM prediction by PhosphoSitePlus . Figure 3: mutation analysis on TCGA PanCancer Atlas Studies through cBioPortal [89, 90]. Figure 4: ZAR1 binding partner Network depiction/analysis by String v11 .
Primers for CoBRA analysis of the ZAR1 promoter (186 bp) were upper primer GGAGAAGGAYGAAGAGGGGTTTTT and lower primer TCCCCCAAAACCRCCATAAAC, and pyrosequencing primer was TGGTAGGAAGGGYGTGGAGG. Primers for RT-PCR were ZAR1 AGCTGGGCAAGGAGCGGCTG and GGTGGGGCCGTTTAGGGTCCA (264 bp), GAPDH TGGAGAAGGCTGGGGCTCAT and GACCTTGGCCAGGGGTGCTA (176 bp), ACTB CCTTCCTTCCTGGGCATGGAGTC and CGGAGTACTTGCGCTCAGGAGGA (226 bp), p21 CCTTGTGCCTCGGTCAGGGGAG and GGCCCTCGCGCTTCCAGGAC (183 bp), p27 GTGCGAGAGAGGCGGTCGTG and TCCACCGGGCCGAAGAGGTT (146 bp), WEE1 CACACGCCCAAGAGTTTGC and CACTTGAGGAGTCTGTCGCA (135 bp) and WEE1 3′UTR primers are: pair 1 CTCCCCCTGAACACTGTGAC and ACTGACACCAATCGAGAAAGT (87 bp), pair 2 CACCAGCCTTTCCAGGGTTA and GGTCACTACAGGGAAAGACACC (92 bp), pair 3 AGCCTTCAATGTACCTGTGTGT and TGCCTACAAAGTGCTCCCAG (93 bp), pair 4 CTGGGAGCACTTTGTAGGCA and AGCAGCAAATTCACAAGGCA (77 bp), pair 5 AGTTTTGTCTTTGCTGTAAACTTGT and CATCAAAAGCAGCTATACATTTCAC (100 bp), pair 6 TGCACCCTTTCCCTCCTTTG and GTCCGGGAAGGACATTACCA (89 bp), pair 7 TGTTTTGCCCGGTTTTTCTCT and GTCAGAAGTCATTCTGGCATTTCA (95 bp), pair 8 TTTGCACTTGTCTTTGACTTGTGT and AGGTAAGCTCAGAGTGACTTTT (70 bp), pair 9 GCCATTTGACTAATAATACTGGCT and ACACAAGTCAAAGACAAGTGC (106 bp).
We thank Sylvia Thomas for preparing the microarray chips, Lienhard Schmitz for plasmids and Reinhard Dammann for support .
VD, JB, SW, and AR performed the experiments. CLIP was performed by OR, mass spectrometry by AS and JG, binding motif prediction by MB, and microarray by TB. AR conceived and designed the experiments and wrote the manuscript. All authors corrected and approved of the submitted manuscript.
TB’s work is supported by the Deutsche Forschungsgemeinschaft DFG GRK2355. OR’s was supported by the Deutsche Forschungsgemeinschaft DFG, Research Training Group (RTG) 2355 (project no. 325443116).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interest.
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