Didych et al. BMC Research Notes 2012, 5:178 http://www.biomedcentral.com/1756-0500/5/178 RESEARCHARTICLE Open Access DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity Dmitry A Didych, Elena S Kotova, Segey B Akopov, Lev G Nikolaev* and Eugene D Sverdlov Abstract Background: Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. Results: Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. Conclusions: We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells. Background Two basic approaches have been proposed to identify Spatial, temporal and tissue specific gene expression in many potential genomic insulators in one experiment. mammals is largely determined by genomic cis-regulatory One approach is based on the ChIP-on-chip or ChIP-seq elements, such as promoters, enhancers, silencers, and techniques with antibodies to known insulator-binding insulators (for recent review, see [1,2]). A survey of about proteins, like CTCF or CP190 [8-10]. This approach can 1% of the human genome [3] indicated that the regulatory be used for the whole-genome analysis, but has a draw- elements were more abundant in the genome than the back that binding of a certain protein may be insufficient genes they control and are mostly distal to the genes that for insulator activity, which may result in many false posi- they regulate. tives. Another approach is based on a functional enhan- While the number and positions of enhancer elements cer-blocking test in stably transfected cells [11,12] but is in the whole human genome can be determined with applicable to only relatively short (several megabases) some certainty through P300 binding [4], the number and genomic sequences. positions of most insulator elements are not known [2], It is well known that most mammalian insulators (with and methods of their identification in mammals are sparse. some exceptions reported [13-15]) bind CTCF (for review, Moreover, the definition of insulator is somewhat ambigu- see [16,17]). However, it was shown that CTCF has many ous–this term designates elements with enhancer-blocking other genomic functions apart from insulator [17]. or chromatin-bordering functions (reviewed in [5]) which Earlier we developed a positive-negative selection are not interrelated at least in some cases [6,7]. In addi- method allowing identification of insulators based on tion, the term “insulator” is sometimes used to designate their ability to prevent promoter activation by enhancer the elements that bind the CTCF protein but have no pro- when located between them [11]. We constructed a ven enhancer-blocking or chromatin-bordering activity. pPNT/EmP plasmid [11,12] containing the neomycin- resistance gene under control of the mouse phosphogly- cerate kinase promoter (mPGK1) and the herpes simplex * Correspondence: [email protected] virus thymidine kinase (HSV-tk) gene under control of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy the CMV minimal promoter and CMV enhancer. HSV-tk of Sciences, 16/10 Miklukho-Maklaya, Moscow 117997, Russia © 2012 Nikolaev et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Didych et al. BMC Research Notes 2012, 5:178 Page 2 of 9 http://www.biomedcentral.com/1756-0500/5/178 catalyzes phosphorylation of ganciclovir to the monopho- was isolated by agarose gel electrophoresis. The sns-con- sphate [18,19] which is further converted into the tripho- taining fragment was inserted in both orientations into sphate by cellular enzymes and incorporated into pPNT/EmP cut with Sal I and treated with Klenow growing DNA chain causing termination of replication enzyme. and cell death [20,21]. A negative control pPNT/E-l-mP plasmid was prepared The pPNT/EmP plasmid efficiently expresses the HSV- by insertion of a lambda phage fragment PCR-amplified tk gene. However, after insertion into pPNT/EmP of a with Xho I site-containing primers ACTCGAGTCCGT- DNA fragment capable of blocking the interaction GAGGTGAATGTG and ACTCGAGTAGTCGGCT- between the CMV promoter and enhancer, the HSV-tk CAACGTGG into SalI-digested pPNT/EmP. expression in cells stably transfected with this plasmid The constructs obtained are shown in Figure 1. Prior gets significantly reduced and the cells become resistant to electroporation, the constructs were linearized with to ganciclovir. Eco47 III (Fermentas). Using this principle, we developed a technique and used To reveal possible silencer activity of CTCF-binding it for identification and mapping of 18 enhancer-blocking fragments, the Sal I recognition site of the pPNT/EmP DNA elements within the FXYD5-COX7A1 region of plasmid was inactivated by Sal I digestion and filling in human chromosome 19. This region contains more than the sticky ends with Klenow enzyme followed by self-liga- 40 characterized genes with different expression profiles, tion. The plasmid was then cut with PspE I (SibEnzyme), and the data obtained allowed us to make conclusions on and double-stranded adapter (a hybrid of GTCAC- the mutual arrangement of enhancer-blocking sequences CAATTGTCGACGGATCC and GTGACGGATCCGTC- and genes and their possible functional interactions GACAATTG) containing a Sal I site (underlined) was [11,12]. inserted 3’ to the HSV-tk gene. The resulting plasmid In this work, we studied the relationship between (pPNT/EmPS, Figure 1) was used for insertion into the CTCF binding and enhancer-blocking activity of 10 novel Sal I site of CTCF-binding fragments ## 3, 7 and 8. CTCF-binding genomic fragments identified earlier in Prior to electroporation, the constructs were linearized our laboratory [22]. Using a functional test described with Ssp I (Fermentas). above, we demonstrated that all fragments which bind CTCF both in vitro and in vivo were capable of blocking Cell culture and transfection activation of the CMV minimal promoter by the CMV CHO-K1 cells (CCL-61, Chinese hamster ovary cells) were enhancer in stably transfected CHO cells. grown under conditions recommended by ATCC. Electro- poration was performed using a Gene Pulser Xcell Methods (BioRad) system as described previously [11], the trans- Basic protocols fected cells were inoculated into 5 ml of growth medium Growth and transformation of E. coli cells, preparation and incubated for 48 hours. The medium was then of plasmid DNA, agarose gel electrophoresis, blot-hybri- replaced with fresh culture medium supplemented with dization and other standard manipulations were per- 500 ug/ml of G418 (Geneticin, Gibco-BRL), and the cells formed as described [23]. were cultured for 2 weeks in the presence of this antibio- tic. An aliquot of the cell suspension was taken for geno- Constructs mic DNA isolation, and residual cells were cultured for Ten in vitro CTCF-binding DNA fragments cloned pre- 2moreweeksafteradditionof4or10uMofGANC viously in pGEM-T (Promega) [22] were cut out with (Sigma). The G418 and GANC resistant cells were then Xho I and inserted in both orientations into pPNT/EmP collected, and genomic DNA isolated using a Wizard [11] using Sal I site located between the CMV enhancer Genomic DNA Purification Kit (Promega). and minimal promoter. A pPNT/mP plasmid containing the HSV-tk gene under PCR control of the CMV minimal promoter and conferring The first stage of nested PCR was performed using a 20 ng resistance to neomycin and ganciclovir on transfected cells genomic DNA template and primers matching the 5’ and [11] was used as one of positive controls. Another positive 3’ flanks of the insertion site (Sal I). Structures of the pri- control was a pPNT/E-sns-mP plasmid containing the sea mers are presented in Table 1. Primers P1L and P1R were urchin Paracentrotus lividus sns insulator between enhan- used for pPNT/EmP based constructs and primers P2L cer and promoter [24,25]. To prepare this construct, a pBS and P2R - for pPNT/EmPS based constructs. The first KS + plasmid, kindly provided by R. Melfi and G. Spinelli stage product was diluted 4-fold and 1 ul thereof was used (University of Palermo, Italy), was cut with HindIIIand as a template for the second stage, where each individual Sma I, treated with Klenow enzyme to fill in the sticky internal primer was used in combination with either P1L ends, and a ~300 bp fragment containing the sns insulator or P1R in order to determine both the presence and Didych et al. BMC Research Notes 2012, 5:178 Page 3 of 9 http://www.biomedcentral.com/1756-0500/5/178 Figure 1 Plasmid constructions used in this work.mPGK-1-mousephosphoglyceratekinaseIpromoter;NeoR-E. coli neomycin phosphotransferase gene; HSV-tk-herpes simplex virus thymidine kinase gene. Broken arrows indicate direction of transcription. For detailed description, see the text. orientation of the CTCF-binding fragments in the selected where C is the relative DNA content, E-efficiency of DNA (see Table 2).