In trans promoter activation by enhancers in transient transfection

Accepted Manuscript In trans promoter activation by enhancers in transient transfection

N.A. Smirnov, S.B. Akopov, D.A. Didych, L.G. Nikolaev PII: DOI: Reference:

S0378-1119(16)30964-7 doi: 10.1016/j.gene.2016.12.005 GENE 41703

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Gene

Received date: Revised date: Accepted date:

25 April 2016 15 November 2016 8 December 2016

Please cite this article as: N.A. Smirnov, S.B. Akopov, D.A. Didych, L.G. Nikolaev , In trans promoter activation by enhancers in transient transfection. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2016), doi: 10.1016/j.gene.2016.12.005

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ACCEPTED MANUSCRIPT In trans promoter activation by enhancers in transient transfection Smirnov N.A., Akopov S.B., Didych D.A., Nikolaev L.G.* Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia *Corresponding author. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian

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Academy of Sciences, 117997, Moscow, Russia. Tel. +7 495 330 7029; fax +7 495 330 6538.

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E-mail [email protected]

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Abstract

Earlier, it was reported that the strong cytomegalovirus enhancer can activate the

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cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-

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reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral

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regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter

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activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers

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shares a number of important traits with long-distance promoter activation by enhancers in

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living cells and may therefore serve as a model of this fundamental cellular process.

Keywords: enhancer-promoter interaction; activation in trans; transient transfection

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ACCEPTED MANUSCRIPT Introduction It is now well established that complex interrelations between genome regulatory elements ultimately determine the functional identity of the cell (for recent reviews, see (Cavalli and Misteli, 2013; Levine et al., 2014)). Using chromosome conformation capture-like (3C-like) techniques of the whole genome analysis (de Wit and de Laat, 2012), direct contacts between

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distantly located promoters and enhancers have been clearly demonstrated. However, alternative mechanisms for long-range enhancer functioning have also been proposed

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(Krivega and Dean, 2012; Kulaeva et al., 2012; Marsman and Horsfield, 2012). Also, despite

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the abundance of the whole genome data on intranuclear contacts, including high resolution human genome 3D maps (Rao et al., 2014), there is still little knowledge on the specific

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mechanisms underlying the formation, maintenance and control of interacting complexes of

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regulatory elements, in particular of the most commonly studied enhancer-promoter complexes. Probably, one of the reasons is that the resolution of the 3C-family assays (>1 kb)

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is still not sufficient, and the fragment under study may include regulatory sequences other

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than promoter and enhancer. Moreover, close proximity of the elements does not necessarily mean functionality (Edelman and Fraser, 2012; van Arensbergen et al., 2014). Another reason is that the mechanisms of enhancer-promoter interactions in the

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genome are complicated, and an active promoter interacts with a number of enhancer

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elements distributed mostly within 500 kb distance from the promoter (Sanyal et al., 2012; Jin et al., 2013; Rao et al., 2014). An enhancer element, in turn, contacts on the average with two promoters, and these elements together with silencers and insulators participate in multiple interactions and form complex networks (Sanyal et al., 2012; van Arensbergen et al., 2014). It is also very difficult to change and/or modify (e.g. methylate) nucleotide sequences of regulatory elements within the genome and chromatin in a living cell. Recently emerged genome editing techniques (for review, see (Nemudryi et al., 2014)) are still too laborious for routine analysis.

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ACCEPTED MANUSCRIPT Therefore, in addition to whole genome approaches, a relatively simple assay of functional long-distance enhancer-promoter interactions that could reflect at least some aspects of the activation mechanism might be of use. Such a model should be relatively independent of the genome context (no position effect) and contain a small number of elements (e.g. one promoter and one enhancer). Using standard DNA manipulation

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techniques, it should also allow to insert/remove/modify short regulatory sequences (like transcription factor binding sites and other elements) within the enhancer and promoter under

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study in order to elucidate their participation in the activation process. In other words, a kind

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of hybrid in vivo and in vitro approaches could be useful to study the long-distance enhancerpromoter activation. In this study we describe a model which meets some of these

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requirements.

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In the late 1980s it was demonstrated that in transient co-transfection assays an enhancer can activate a promoter located on a different plasmid when these elements are kept

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together by non-covalent bonds (Dunaway and Droge, 1989; Mueller-Storm et al., 1989).

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Later it was shown that the effect of activation in trans can be observed at co-transfection of a plasmid containing enhancer and a plasmid containing a promoter and a reporter gene, i.e. under penetration of multiple copies of the promoter- and enhancer-containing plasmids into

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the cell. In such an experiment the strong cytomegalovirus enhancer activated the

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cytomegalovirus promoter in trans (i.e. when located on a separate plasmid co-transfected with a promoter-reporter gene construct) (D'Aiuto et al., 2006). In the present study, we aimed to further investigate the promoter and cell-type specificity of the in trans promoter activation by enhancers, and to initially characterize the mechanism of this activation. We demonstrate that the ability to activate promoter in trans in transient transfection experiments is a property of not only viral enhancers and promoters but also of genomic elements, and the activation is accompanied by physical interaction between promoter and enhancer as revealed by the 3C assay. We believe that the co-

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ACCEPTED MANUSCRIPT transfection/reporter gene assay of the enhancer activity in trans might be a useful model for studying many important properties of long-distance enhancer-promoter communications including cell type specificity, enhancer-promoter specificity etc.

Materials and Methods

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Plasmid constructs For the co-transfection experiments, three promoter plasmids (Pu2p, Pcmv and Ptk), five

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enhancer plasmids (E2, E4, E6, Esv40 and Ecmv), and one control plasmid L, containing

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neutral lambda-DNA, were prepared.

Pu2p plasmid was obtained by insertion of the human genomic bidirectional U2P

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promoter of the U2AF1L4 and PSENEN genes (Didych et al., 2013) into a pGL4.10 vector

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(Promega) upstream of the firefly luciferase gene, as described previously. The orientation of the promoter corresponded to the U2AF1L4 gene transcription.

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Pcmv plasmid, containing the Photinus pyralis (firefly) luciferase gene under the

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control of a minimal 61 bp cytomegalovirus (CMV) promoter, was prepared by excising a 318 bp fragment containing a minimal CMV promoter and the hTERT promoter from pGL3hTERT-CMV plasmid (Kuzmich et al., 2014) with Xho I and Bgl II, and cloning it into

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pGL4.10 plasmid upstream of the firefly luciferase gene. The hTERT promoter was then

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excised from the construct by digestion with Xho I and Eco RI, and the plasmid was closed by filling in its sticky ends using Klenow enzyme and a blunt-end ligation. The Ptk promoter was tested as a part of pRL-TK plasmid (Promega) containing the Renilla reniformis luciferase gene under the control of the HSV-tk gene promoter. This plasmid is commonly used as a reference in the Promega dual-luciferase reporter assay system (Allard and Kopish, 2008) to correct for variations in transfection efficiency between samples. However, since our experimental system aimed to identify the action of an enhancer on a promoter in trans, we used the activity of the Renilla luciferase as a measure of the Ptk

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ACCEPTED MANUSCRIPT promoter activity. We did this, rather than taking the dual luciferase approach, because certain sequences in the test plasmid may affect regulatory elements of the reference plasmid leading to experimental artifacts, as noted in (Farr and Roman, 1992; Adam et al., 1996). The human genome fragments E2, E4 and E6, which, as we demonstrated previously, are active enhancers in HeLa and HepG2 cells (Smirnov et al., 2013), were inserted into a

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pAL-TA vector (Evrogen), which resulted in plasmids E2, E4 and E6, respectively. To obtain Esv40 plasmid, the SV40 enhancer was amplified with GAAGGAGCTGACTGGGTTGA and

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AAAGCAAGTAAAACCTCTACAAATGTG primers on the pGL4.13 plasmid (Promega)

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template and cloned into the pAL-TA vector (Evrogen). A DNA fragment containing the CMV minimal promoter was excised from pPNT/EmP plasmid (Akopov et al., 2006) with

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Xho I and Hind III, its 5’-ends were filled in with the T4 DNA polymerase, and poly-T tails

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were added with the terminal nucleotide transferase. The resulting fragment was gel-purified and cloned into the pAL-TA vector to form Ecmv plasmid.

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A control plasmid (L) was obtained by PCR-amplification of a 162 bp fragment on

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lambda phage DNA template using TCCGTGAGGTGAATGTGGTG and TAGTCGGCTCAACGTGGGTT primers and cloning the fragment into the pAL-TA vector

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(Evrogen).

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Transfection and reporter gene assay HeLa (human epithelial cervical carcinoma, ATCC-CCL-2), A431 (human epidermoid carcinoma, ATCC-CRL-1555) and HepG2 (human hepatocellular carcinoma, ATCC-TIB152) cells were grown at 37°C and 5% CO2 in DMEM/F12 (1:1) medium containing 10% fetal calf serum. The plasmid DNA mixture for each transfection was prepared in 20 ul of TE buffer from 40 ng of the promoter plasmid, 75 ng of pRL-TK plasmid (Promega), 3-10-fold molar excess of the enhancer plasmid (enhancer-promoter ratio 3-10), and adjusted to 500 ng of total

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ACCEPTED MANUSCRIPT DNA with the control plasmid L so that the total amount of transfected DNA in each transfection was the same. Transfections were performed in 24-well plates at 100-200x103 cells per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Each well was incubated with 500 ng of mixed plasmid DNA for 18 h, after which the cells were lyzed in

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PLB buffer (Promega), and the activities of the Photinus pyralis and Renilla reniformis luciferases were assessed separately using a GENios Pro luminometer (Tecan) and the Dual

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Luciferase Reporter Assay System (Promega). The activation of the promoter was calculated

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as the activity of the corresponding luciferase under the control of the promoter in the presence of the enhancer plasmid normalized to the activity of the same plasmid in the

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presence of the control enhancer-less plasmid L. Three independent transfections with two

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parallel measurements of each luciferase activity were carried out, and the standard error of

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Enhancer-promoter interaction

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the mean calculated.

A chromosome conformation capture assay with a quantitative PCR analysis was performed as described (Hagege et al., 2007). Briefly, about 106 HepG2 cells co-transfected for 24 h with

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10 ug of an equimolar mixture of Pcmv, Ecmv and L plasmids were cross-linked with 1%

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formaldehyde for 10 min at room temperature. The reaction was quenched with 2 M glycine, and the cells were lyzed with 5 ml of NP-40 buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.15% (v/v) NP-40, 0.15 mM AEBSF), washed and resuspended in the same buffer without NP-40. Up to 0.3% (w/v) SDS was then added to the nuclei, and the suspension incubated at 37°C for 1 h. To sequester SDS, Triton X-100 was added to a final concentration of 2.5% (v/v), and the suspension was further incubated at 37°C for 1 h. The nuclei were then digested with Msp I in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT (NEB2 buffer), followed by ligation as described in (Hagege et al., 2007), and treated with 30 ug/ml

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ACCEPTED MANUSCRIPT RNase A for 30 min at 37°C and 30 ug/ml proteinase K at 65°C overnight to reverse crosslinks. DNA was extracted with phenol-chloroform, ethanol precipitated, and used for realtime PCR amplification. The Msp I digestion efficiency was measured for all plasmids as described (Hagege et al., 2007) to be in all cases higher than 85% (Table 1).

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Negative (no cross-linking) control was done at the conditions as above but without the addition of formaldehyde.

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qPCR reactions were performed using a qPCRmix-HS SYBR system (Evrogen) and

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the Lightcycler 480 (Roche, USA) in accordance with the manufacturers’ instructions. DNA fragments were PCR-amplified for 40 cycles at 95oC for 20 s, 63oC for 30 s, 72oC for 20 s.

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The amplified DNA fragments were additionally analyzed for their homogeneity by a 1.2%

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agarose gel electrophoresis and by melting curve analysis. A single PCR product of the expected length (351 and 430 bp for promoter-control and promoter-enhancer ligation

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product, respectively) was detected in all reactions.

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A random ligation template (Splinter et al., 2004) was prepared by digestion of an equimolar mixture of Pcmv, Ecmv and L plasmids with Msp I and ligation of the obtained fragments at high DNA concentration. The PCR efficiency of primer pairs was determined by

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amplification of serial dilutions of the template and calculated using the LinRegPCR program

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(Ruijter et al., 2009).

The relative content of the enhancer-promoter and promoter-control ligation products was determined by a real-time PCR assay as follows. The number of cycles necessary for amplification of the ligation products from the random ligation template (RLT) to the threshold value was plotted against serial dilutions of RLT. Using the logarithmic trendline of this plot (R2>0.97), the effective dilutions corresponding to the promoter-control and enhancer-promoter ligation products were determined, and the relative content (Cr) of each product calculated:

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Cr = 1000/D, where D is the effective dilution.

Two independent 3C experiments were performed and standard error of the mean calculated. No ligation products were detected even after 40 cycles of real-time PCR when the

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formaldehyde cross-linking step was omitted.

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Nuclear penetration of the transfected plasmids

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HepG2 cells were transfected with an equimolar mixture of Pcmv, Ecmv and L plasmids, cross-linked, and nuclei isolated using NP-40 as described above (section

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Enhancer-promoter interaction). Nuclear DNA was then extracted with phenol-chloroform,

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precipitated with ethanol and used as a template for real-time PCR with primers specific for plasmids Ecmv and L (plasmid control primers shown in Table 1). The equimolar mixture of

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the plasmids was used as a control template. Normalized amounts (XN) of each plasmid were

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calculated as:

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XN=(1+E)-dCt,

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where E is the PCR efficiency (see Table 1), and dCt is the difference between the threshold number of cycles for the amplification of nuclear DNA and the control mixture.

Results Enhancer-promoter activation in trans We co-transfected HepG2 cells with the HSV-tk promoter (Ptk) plasmid and several enhancers of different origin. The results are shown in Fig. 1A. As seen from the figure, the most pronounced activation (>10-fold) was observed in the case of co-transfection of the

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ACCEPTED MANUSCRIPT constructs containing the CMV enhancer and the HSV-tk promoter at 10:1 enhancer-promoter ratios. However, other enhancers, including the viral SV40 enhancer and the three human genome enhancers tested, were also able to 2-5 fold activate the HSV-tk promoter in HepG2 cells. Also, it can be seen that the transactivation degree clearly depends on the enhancerpromoter ratio.

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This effect is presented in more detail in Fig. 1B. As seen, the higher the enhancerpromoter ratio, the higher the activation degree, and in HeLa cells this degree tends to plateau

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at the enhancer-promoter ratio about four.

Cell-type and promoter specificity

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The enhancer-promoter activation in trans during transient transfection revealed a

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considerable degree of specificity to the promoter in HepG2 cells (Fig. 2). In particular, the viral Ecmv and Esv40 enhancers much better activated in trans both viral promoters (Pcmv

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and Ptk) than cellular Pu2p promoter (Fig. 2C). At the same time, cellular enhancers E2 and

in HeLa cells (Fig. 2B).

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E4 had substantially lower promoter specificity. This feature of enhancers was also observed

The activity in trans of the enhancers tested was evidently cell type-specific (Fig. 2). It

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was maximal in hepatocellular carcinoma (HepG2) cells, lower in cervical adenocarcinoma

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(HeLa) cells and virtually not detected in epidermoid carcinoma cells (A431) under the conditions used.

Enhancer-promoter interaction The mechanism of long (genomic) distance enhancer-promoter communications which suggests a close contact between enhancer and cognate promoter sequences is probably the most widely accepted to date (reviewed in (Levine et al., 2014)). We applied the original 3C approach with real-time PCR analysis (Hagege et al., 2007) to reveal whether regulatory 9

ACCEPTED MANUSCRIPT elements located in different plasmids are brought to contact when an enhancer activates a promoter in trans. To this end, we co-transfected HepG2 cells with equimolar amounts of three plasmids: a plasmid containing the firefly luciferase reporter gene under the control of a CMV minimal promoter (Pcmv), a plasmid containing the CMV enhancer (Ecmv) and a plasmid (L) containing a similar in length neutral fragment of phage lambda DNA (Fig. 3A).

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After cross-linking, DNA purification and ligation (see Methods), the ligation frequency of the enhancer-promoter and promoter-control was determined by quantitative PCR with

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primers (Table 1) targeted at the corresponding ligation product. The results are presented in

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Fig. 3. Fig. 3B shows a schematic representation of the putative complexes detected with the corresponding primer pairs. Fig. 3C presents the relative content (see Methods) of the Pcmv-

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Ecmv and Pcmv-L ligation products shown as an average of two independent transfection-3C

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experiments.

As seen from the figure, the DNA content (and the corresponding ligation frequency)

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of Pcmv and Ecmv was more than 50-fold higher than that of Pcmv and the control lambda

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sequence. These data show that the CMV promoter and enhancer are specifically brought together when co-transfected into HepG2 cells. No Pcmv-Ecmv or Pcmv-L ligation products were detected in the non-cross-linked control.

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In order to eliminate the possibility of artifacts due to different efficiency of

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penetration of enhancer and control plasmids into the cell nucleus, we compared the relative quantities of Ecmv and L DNAs in HepG2 cell nuclei after transfection (see Methods for detail). The normalized amounts XN (see Methods) were calculated to be 0.53 and 0.50 for Ecmv and L plasmid, respectively, indicating that both constructs penetrate the nucleus with almost equal efficiency.

Discussion

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ACCEPTED MANUSCRIPT We designed a system that could reliably and at least semi-quantitatively detect the enhancer-promoter activation in trans, and reflect general properties of long distance enhancer-promoter activation, such as tissue and promoter specificity. Since this system is based on transiently transfected constructs and the reporter gene assay, it is independent of the genome context.

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There are very few reports regarding enhancer-promoter activation in trans in transient transfection. It can be partially explained by the fact that in the standard reporter gene assay

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with a reference plasmid (like in the dual luciferase assay), the promoter activation in trans

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will be masked by simultaneous influence of the enhancer on both experimental and reference promoters. This is the reason why the most detailed to date study of enhancer-promoter

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activation in trans after co-transfection was performed using flow cytometry and not a

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reporter gene assay (D'Aiuto et al., 2006). To avoid this bias, in our transfection/reporter gene experiments we did not use normalization to a reference gene, instead we assumed that the

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transfection efficiency in a single experiment was the same for all transfection samples. This

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assumption is supported by a relatively small standard error of the mean values obtained in our measurements of relative luciferase activity (see Figs. 1 and 2). The activity in trans is a rather common phenomenon not limited to a small number of

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cell types studied here. As reported previously (D'Aiuto et al., 2006), the viral CMV enhancer

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is also active in trans in HEK-293 and CHO-K1 cells. Here, we demonstrated that the ability to activate promoter in trans in a transient transfection assay was not limited to viral enhancers as shown earlier (D'Aiuto et al., 2006), but it is also characteristic of genomic regulatory elements. Enhancer-promoter activation in trans is promoter- and cell type specific and includes a physical contact of promoter and enhancer as revealed by the 3C assay. Therefore, the promoter activation by an enhancer in trans reflects some basic properties of the enhancer-promoter activation in native cells and may be used as a model to study long-distance enhancer-promoter activation mechanisms.

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ACCEPTED MANUSCRIPT Moreover, this approach is advantageous in the possibility to easily prepare and test mutated promoter and enhancer sequences which allows to elucidate the role of different transcription factors binding sites and quantitatively measure the influence of chromatin structure and other regulatory elements, like insulators, on the effect of activation. Of course, the proposed model has some drawbacks. The main one is that chromatin

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structure and histone modifications of the transfected plasmid are not known in sufficient detail. As shown previously, transfected plasmids are chromatinized, although with a lower

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histone H1 content and some special features in positioning of nucleosomes (Hebbar and

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Archer, 2008). A lower H1 content may be characteristic of actively transcribed chromatin where H1 is partially replaced with other proteins, in particular with high mobility group

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proteins (for review see (Thomas and Stott, 2012)). However, additional study is necessary to

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take all the advantage of this model.

Also, at the standard lipofection conditions, the number of exogenous enhancers and

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promoters introduced into the cell (several thousand molecules, see (Cohen et al., 2009)) is

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much higher than that of any single type of cellular promoters or enhancers, with a possible exception of the promoters/enhancers that are parts of repeated elements. A great number of exogenous regulatory elements may bind certain transcription factors necessary for enhancer-

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promoter activation and thus interfere with the cellular regulatory network, which should be

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taken into account in the interpretation of the results. This effect might, for example, explain the cell type specificity of enhancer-promoter activation found in our experiments, if to suppose that transcription factors required for the activation are less abundant in HeLa and A431 cells as compared with HepG2. Our results also indicate that in transient transfection, when multiple plasmid copies are transfected into a single cell, the activation mechanism may also be realized when enhancer of one plasmid activates in trans promoter on another plasmid. Moreover, the data presented here suggest that the influence in trans of transfected enhancers (and, possibly,

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ACCEPTED MANUSCRIPT promoters and other regulatory elements) on the transcription of the host cell genes also should be taken into account in the interpretation of transient transfection experiments. Summarizing, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular

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process.

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Acknowledgement

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The authors thank B. O. Glotov for critical reading of the manuscript and valuable comments, and E.D. Sverdlov for helpful discussion. This work was supported by the grant program for

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the leading scientific schools of Russia (Project NSh_1674.2012.4) and by the Russian

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Academy of Sciences Molecular and Cellular Biology program.

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ACCEPTED MANUSCRIPT Legends to figures Fig. 1. Enhancer-promoter activation in trans. Enhancer activity was normalized to that of a lambda DNA fragment negative control (L). (A) Activation of the thymidine kinase promoter (Ptk) of pRL-TK plasmid in HepG2 cells by different co-transfected enhancers (described in the text). L – a lambda DNA fragment negative control. 1:3, 1:10 – enhancer-promoter molar

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ratio. (B) Effect of the enhancer-promoter ratio on the activation of the Pcmv promoter by the

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Ecmv enhancer co-transfected into HeLa cells.

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Fig. 2. Cell type and promoter specificity of enhancer-promoter activation in trans. L – a lambda DNA fragment negative control. 1:3, 1:10 – enhancer-promoter molar ratio. (See the

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text for description of the promoters and enhancers).

Fig. 3. Proximity of the regulatory elements during enhancer-promoter activation in trans as

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revealed by a chromosome conformation capture (3C) assay in HepG2 cells. (A) – schematic

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representation of the co-transfected plasmids; (B) - putative complexes detected with the corresponding primer pairs; (C) - the relative content of the ligation products. Pcmv, Ecmv – the CMV promoter and enhancer, respectively. L – a lambda DNA fragment used as negative

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control. LUC – the firefly luciferase reporter gene. Relative DNA content reflects the ligation

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frequency of the corresponding regulatory elements.

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ACCEPTED MANUSCRIPT

Table 1. Digestion efficiency and PCR efficiency tests. Plasmid

Primers flanking Msp I site

Ecmv 1

GTGTATCATATGCCAAGTACGC

Digestion, %

Сontrol primers

E*

97.5 GTTTTCCCAGTCACGACGTTG

GTGTATCATATGCCAAGTACGC

GGCTATGAACTAATGACCCCGTA

GTGAGTCAAACCGCTATCCAC

0.94 Ecmv 2

98.1

Lambda

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CACGACAGGTTTCCCGACT ATAGAGCATAAGCAGCGCAAC

AGCTATGACCATGATTACGCCAA

93.9 CTCCCACCGTACACGCCTA

96.8 CGATAGTACTAACATACGCTCT

TACATGAGCACGACCCGAAAGCC

0.935

CGCACCGCTTGTGTCCGATT

TCCCGTCTTCGAGTGGGTA

85.9

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Pcmv 2

SC

Pcmv 1

0.935

ATAGAGCATAAGCAGCGCAAC

RI

CACGACAGGTTTCCCGACT

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CGCTAGCCTCGAAATTCGGTA

AC

CE

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D

*PCR efficiency of control primer pairs;

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RI

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ACCEPTED MANUSCRIPT

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Figure 1

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ACCEPTED MANUSCRIPT

Figure 2 20

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ACCEPTED MANUSCRIPT

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RI

Figure 3

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ACCEPTED MANUSCRIPT

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Abbreviations CMV – cytomegalovirus, HSV-tk – herpes simplex virus thymidine kinase, RLT – random ligation template, 3C – chromosome conformation capture, Ptk – herpes simplex virus thymidine kinase promoter

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ACCEPTED MANUSCRIPT Highlights • Transfected enhancers are capable to activate promoters located on separate plasmids. • This enhancer/promoter activation in trans is promoter- and cell type specific.

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• Activation is accompanied by physical interaction between promoter and enhancer.

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