ISSN 0006-2979, Biochemistry (Moscow), 2013, Vol. 78, No. 8, pp. 895-903. © Pleiades Publishing, Ltd., 2013.
Published in Russian in Biokhimiya, 2013, Vol. 78, No. 8, pp. 1141-1150.
Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM13-087, June 23, 2013.

Assay of Insulator Enhancer-Blocking Activity
with the Use of Transient Transfection
N. A. Smirnov, D. A. Didych, S. B. Akopov*, L. G. Nikolaev, and E. D. Sverdlov
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10,
117997 Moscow, Russia; fax: (495) 330-6538; E-mail: akser@ibch.ru
Received March 25, 2013
Abstract—We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selectedfrom the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity whenprobed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity andby dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used forquantitative analysis of the enhancer-blocking activity of the potential insulators.
DOI: 10.1134/S0006297913080051
Key words: human genome, gene expression regulation, insulator, enhancer

Insulators are DNA sequences that prevent activation of promoters by inappropriate enhancers and/or
block the spread of condensed chromatin (for a recent
review see [1]). Insulators have been identified in various
eukaryotic organisms, including vertebrates, Drosophila,
sea urchin, and yeast [2-6]. Some insulators can function
when transferred into phylogenetically distant organisms,
like sea urchin, plants, and human [5, 7], and can interfere with heterologous enhancers [7, 8].
The activity of different insulators can depend [2, 9,10] or not depend [11-13] on their orientation relative tothe cognate promoters. If more than one insulator is
located between enhancer and promoter, their combined
enhancer-blocking activity can be much lower than thatof a single insulator [14-16]. This neutralization effectcan depend on the orientation of insulators relative toeach other [17]. Consequently, insulators may not justsubdivide a genome into domains but rather form, incooperation with genes, promoters, enhancers, and otherelements, a multilevel network regulating the transcriptional activity of the genome.
Functioning of many insulators depends on bindingof transcription factor CTCF (reviewed in [18, 19]).
However, it would be incorrect to consider all CTCFbinding sequences as insulators.
Quantitative characteristics of the insulator activity
are not sufficiently studied. In most cases the activity of* To whom correspondence should be addressed.

insulator is estimated by its ability to block enhancer–
promoter interactions (enhancer-blocking insulators).
There exist just a few general methods to analyze insulator activity and several methods appropriate only for specific organisms, like the assay developed by Kellum and
Schedl [20] and other similar assays, which are applicable
only for Drosophila [21-23].
Among general methods, the most frequently used
are the following: 1) the approach of Chung et al. [3],which allows for the quantitation of the insulator activity.
This approach is based on insulation of the NeoR gene byflanking sequences, which leads to death of cells whosegenome contains constructs with insulators; 2) the negative–positive selection approach [24]. This approach, incontrast to the previous method, is based on survival of
the insulator-containing cells, so their DNA can be isolated and analyzed. It also allows for the quantitation of
the insulator activity [13]; 3) the transient transfection
assay using constructs expressing the luciferase or GFP
reporter genes and containing insulator between
enhancer and promoter. Comparison of expression of thereporter gene by this construction and similar construction containing the same insulator positioned outside of
the promoter–enhancer pair allows for estimation of theinsulator’s enhancer-blocking activity. To avoid interaction of enhancer and promoter along the circular plasmid, these elements are either separated by two potential
insulators [25] or the measurements are performed using
linearized plasmid [26].

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This last method is the simplest and fastest of all
mentioned. However, it is also the most artificial and is
not allowed for chromatin structure and genome neighborhood on the regulatory elements. Despite this, it is
widely used for evaluation of the insulator activity (forrecent examples see [27-29]). Here we used the linearized
plasmid transfection approach to check for enhancerblocking and other activities of several potential insulators identified by different methods.

MATERIALS AND METHODS
Basic protocols. Growth and transformation of E.
coli cells, preparation of plasmid DNA, agarose gel electrophoresis, and other standard manipulations were performed as described [30].
Cell culture. HeLa (human epithelial cervical carcinoma), HepG2 (human hepatocellular carcinoma), andCHO (Chinese hamster ovary) cells were grown at 37°C
and 5% CO2 in DMEM/F12 (1 : 1) medium containing
10% fetal calf serum.
Plasmid constructions. To construct the pGL4PV
plasmid containing the luciferase gene under control of
SV40 minimal promoter, the 215-bp promoter fragment
was cut out from pGL3-Promoter Vector (Promega,
USA) with XhoI and HindIII and inserted into pGL4.10
plasmid (Promega) cut with the same enzymes.
Plasmid pGL4EPV, containing SV40 minimal promoter and enhancer, was made from the pGL4PV plasmid by digestion of the latter with XbaI and BamHI andligation with the enhancer fragment obtained frompGL3-Control Vector (Promega) by digestion with XbaIand BamHI. pGL4EPV was used for cloning of thepotential insulators into the unique SalI site located outside of the promoter–enhancer pair.
To obtain the pGL4EPV2 plasmid, the pGL4PV plasmid was linearized by SalI digestion, and the resulting ends
were filled-in with Klenow enzyme. Next, the 353-bp
enhancer fragment was cut out from pGL3-Control Vector
with HpaI and BamHI, blunt-ended with Klenow enzyme,
purified by electrophoresis in 1.5% agarose gel, and ligatedwith the linearized pGL4PV. The pGL4EPV2 plasmid wasused for cloning of the potential insulators into the unique
BamHI site located between SV40 promoter and enhancer.
Constructions for enhancer activity assay. Six DNA
fragments containing potential enhancers and five fragments containing potential insulators were selected fromthe human genome (see “Results and Discussion” fordetail), PCR-amplified using human genome DNA as atemplate (structure of the primers are presented in Table1), and cloned into pGEM-T plasmid (Promega). The
fragments were next cut out from the plasmids with PstI
and Cfr42I (isoschizomer of SacII) and, after filling-in of
the sticky ends, ligated into pGL4PV cut with SalI andtreated with Klenow enzyme.

Constructions for enhancer-blocking assay. As a standard enhancer-blocking element, we employed the wellcharacterized cHS4 insulator from chicken β-globin
locus [31]. The pJC13-1 plasmid (kindly provided by
Gaelle M. Lefevre and Gary Felsenfeld, NIDDK,National Institutes of Health, USA) was digested with
KpnI, treated with Klenow enzyme to fill-in the ends, and1.2-kb fragment was purified by electrophoresis in 1.5%
agarose gel. The fragment was cloned in both orientations
into pGL4EPV and pGL4EPV2 previously cut with correspondingly SalI and BamHI and treated with Klenow
enzyme to fill-in the ends. Orientation of the insert relative to SV40 promoter was determined by PCR-amplification with the insert-specific primer and primer complementary to the plasmid sequence.
Phage λ fragment PCR-amplified using phage
genome DNA template and primers TCCGTGAGGTGAATGTGGTG and TAGTCGGCTCAACGTGGGTT
was used as a negative control. The fragment was first ligated into pGEM-T Easy vector (Promega), cut out with
EcoRI, and made blunt-ended with Klenow enzyme.
Next, the insert (~200 bp) was purified by electrophoresis
in 1.5% agarose gel and ligated into pGL4EPV andpGL4EPV2 plasmids as described above for the cHS4
insulator.
The potential insulators, which we identified earlier
and cloned in pGEM-T Easy vector [24, 32, 33], were cut
out with SalI (its recognition site located in primer
sequence flanking the insert [24]), isolated, and cloned
into pGL4EPV and pGL4EPV2 plasmids in both orientations, as described above for the cHS4 insulator.
Before transfection, the plasmids were linearizedwith PstI and purified from the traces of non-digested
DNA in agarose gel.
Transfection and luciferase activity measurement.
Transfection was performed in 24-well plates using
Lipofectamine 2000 (Invitrogen, USA) according to the
manufacturer’s protocol. For transfection, 200 ng of linearized plasmid DNA and 75 ng of reference pRL-TK
plasmid (Promega) were added to each well. After 48-hincubation, the cells were lysed in PLB buffer (Promega),
and the activity of Photinus pyralis and Renilla reniformis
luciferases was assessed using a GENios Pro luminometer(Tecan) and Dual-Luciferase Reporter Assay System(Promega). Firefly luciferase activity was normalized tothat of the Renilla luciferase. Three or four independenttransfections with two parallel measurements of eachluciferase activity were carried out, and the standard errorof the mean was calculated.

RESULTS AND DISCUSSION
Selection of DNA fragments. Based on the
ENCODE project ChIP-seq data as presented in the
UCSC Human Genome Browser [34], we selected six
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ASSAY OF INSULATOR ENHANCER-BLOCKING ACTIVITY WITH TRANSIENT TRANSFECTION
characteristic DNA fragments with potential enhancer
and five fragments with potential insulator properties.
The selection was made based on the ability of the fragments to bind transcription factors p300 and CTCF, theirchromatin DNase I hypersensitivity, and on the state of
their chromatin structure determined by nine epigenetic
markers including CTCF binding and different types ofhistone modification [35, 36]. The fragments defined in[35, 36] as “strong enhancers” and “insulators” wereselected. Positions in the human genome and propertiesof the selected fragments are summarized in Table 1.
In addition, we selected four DNA fragments previously characterized experimentally as enhancer-blockers
by means of positive–negative selection assay [24, 33].
The properties of these fragments are summarized inTable 2.

897

Enhancer activity of genome fragments. To test for
their enhancer activity, the DNA fragments selected fromthe human genome were PCR-amplified on the genome
DNA template (primer structures are presented in Table1), cloned into pGL4PV plasmid downstream to theluciferase reporter gene (Fig. 1), and transfected into
HeLa and HepG2 cells. This transfection was performed
using circular plasmids, and all other transfections withlinearized plasmids.
Of six fragments selected as potential enhancers (E1E6, see Fig. 1 and Table 1), three were able to activate
SV40 promoter under the conditions used in HepG2
cells, and only one (E2) was active also in HeLa cells. Of
five fragments selected as potential insulators (CTCF
binders), one (C3) demonstrated relatively low enhancer
activity in HepG2 cells. Other fragments, as expected,

Table 1. Properties of genomic fragments selected by their functional characteristics
Fragment
ID

Position onhuman chromosome 19(assembly
hg19)

Length,bp

Chromatinstate*,**

CTCF
binding*

p300
binding*

DNase I
hypersensitivity*

E1

3570465935705401

GTGGACCGTGTAGCCGAGAGACAGCCCAGCAAGCTCA

743

strongenhancer GK

–

+++
GH

+++
AGH

E2

3580967135810383

GACAGTGAGCACAGGCTATCACTCTACCCCCGGCCAGTC

713

strongenhancer KP

–

–

+++
AHK

E3

3587110535872127

TGCTTACAGCTTTTCCAGCTAAAAGGAGAGCCAGCATCAGTC

1023

strongenhancer KP

–

+++
K

+++
K

E4

3587940635880078

GCTGGCTGTCCTCATTATTCCGAAGGATTTTTCTTGCCCACT

673

strongenhancer KP

–

+++
K

+
HKP

E5

3594957535950799

ACACTCCCACCAGGAATCAGCCGTGGAAACTCAAATGG

1225

strongenhancer GP

–

+++
G

++
AGP

E6

3661082036611414

CTCCAGCAGTCCCTTGTGTCCATCCAAGCTCCCAGAGG

595

strongenhancer/promoter P

–

+++
P

++
P

C2

3608310936083439

CACTATGATGGTGAAAGCAGTTTGTTAAGAGAAAGGTAAGTGA

331

insulator
GKP

+++
IKG

–

++
AGH

C3

3591126835911580

CACGTCTTCCATCAAGGACTCACACTCTGCGGTTCACAC

313

strongenhancer G

+++
GK

–

++
AGHP

C4

3595962035960321

TGAACTCTGCCCTAAGACCTGTTTCAGACCTCTTTCCGTA

702

insulator
KP

+++
IKG

–

++
AGHK

C6

3618465336185486

GATTCCGGGATAAATGACACACTAGACTCAAGAAGGCACT

834

insulator
GKP

+++
GK

++
K

+++
AGHK

C8

3637273336373330

TTAATACTCCAAGGTTGTCCACACGACCTATATTTACAGT

598

insulator
GKP

+++
GKI

–

+++
AH

Primers* Letters in the corresponding cells indicate the cell lines where this activity was detected: H – HeLa, G – GM12878, P – HepG2, K – K562,
I – IMR90, A – A549. The binding efficiency was estimated basing on the ENCODE ChIP-seq data [40]: from (–) – no binding to (+++) –strong binding.
** Chromatin state was defined by nine epigenetic markers, including binding of CTCF and different types of histone modifications. The fragments
referred by the authors of [35, 36] as “strong enhancer” or “insulator” types were selected.

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Table 2. Properties of potential insulators identified by positive–negative selection
Fragment ID

Position on human chromosome 19 (assembly hg19)

Length,bp

Ins136547955-36548158

204

Ins2

36033501-36033722

Ins3
Ins4

Chromatin state*,**CTCF
binding*

p300
binding*

weak enhancer (GP)transcribed (K)

+
K

–

222

weak enhancer (G)transcribed (KP)

++
G

–

36034844-36035321

478

weak enhancer/weak promoter (GKP)

+
K

+/–
K

35668221-35668887

667

weak enhancer/
transcribed (K)––* Letters in the corresponding cells indicate the cell lines where this activity was detected: H – HeLa, G – GM12878, P – HepG2, K – K562,
I – IMR90, A – A549. The binding efficiency was estimated basing on the ENCODE ChIP-seq data [40]: from (–) – no binding to (+++) –strong binding.
** Chromatin state was defined by nine epigenetic markers, including binding of CTCF and different types of histone modifications [35, 36].

were not active as enhancers. It should be noted that fragment C3, except for binding CTCF, had also properties ofpotential enhancer (see Table 1).
Therefore, four out of seven (60%) of the potential
enhancers demonstrated the expected activity in the system employed in this study, indicating significant (but notcomplete) correlation between the properties used fortheir selection and their enhancer activity.
Enhancer-blocking activity of genome fragments. To
check for the ability of the selected DNA fragments toblock the promoter activation by nearby enhancer, wecloned these fragments between SV40 promoter andenhancer. Control constructions containing the samefragments downstream from the enhancer (outside of thepromoter–enhancer pair, schematically presented in Fig.
2a) allowed assaying silencer activity of the fragments,
which could interfere with the enhancer-blocking activity assay. The constructed plasmids were linearized, transfected into mammalian cells, and luciferase reporter geneactivity was assessed using the Dual-Luciferase Reporter
Assay System (Promega).
Four out of five (80%) genome fragments selected as
potential insulators (see Table 1) showed evident ability to
reduce the extent of activation of SV40 promoter by SV40
enhancer in HeLa cells only when placed between theseelements (Fig. 2a). A possible exception is fragment C4,

Fig. 1. Enhancer activity in HeLa and HepG2 cells of DNA fragments selected from the human genome. The fragments were selected as apotential enhancer (E1-E6) or potential insulators (C2-C8) (for their properties, see Table 1). The selection was made according to the criteria indicated in the “Results and Discussion” section. The DNA fragments were cloned into pGL4PV plasmid as shown in the figure (top).
Circular plasmids were used for the luciferase activity assay. Control plasmids: PV – pGL4PV (contains SV40 promoter and no enhancer);
EPV – pGL4EPV (contains SV40 promoter and enhancer). The activity was normalized to that of pGL4PV.

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ASSAY OF INSULATOR ENHANCER-BLOCKING ACTIVITY WITH TRANSIENT TRANSFECTION

899

a

b

Fig. 2. Enhancer-blocking activity in HeLa cells of DNA fragments from the human genome. a) The DNA fragments were cloned either into
the pGL4EPV2 plasmid between enhancer and promoter (dark columns) or into pGL4EPV plasmid outside of the promoter–enhancer pair(light columns) in both orientations. “+”, orientation of the fragment coincide with its orientation in the genome; “–”, orientation of thefragment is opposite to its orientation in the genome. Linearized plasmids PV and EPV were used for the transfection (see legend to Fig. 1).
The luciferase activity was normalized to that of the pGL4EPV. b) Alignment of the CTCF binding sites found in the studied DNA fragments.
In the right column, the extents of the enhancer blocking by the corresponding fragments in both orientations relative to promoter are shown.
Nucleotides that coincide with the consensus sequence (shown as a Logo) are underlined.

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abFig. 3. Enhancer-blocking activity of the cHS4 insulator from chicken β-globin locus with respect to SV40 promoter–SV40 enhancer (a) andSV40 promoter–E2 enhancer (b) combinations. The luciferase activity was normalized to that of pGL4PV.

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ASSAY OF INSULATOR ENHANCER-BLOCKING ACTIVITY WITH TRANSIENT TRANSFECTION
the enhancer-blocking activity of which was not detected
despite strong binding of CTCF and chromatin state typical for insulators (Table 1). It was reported that CTCF
binding is not necessary for a sequence to display
enhancer-blocking activity [37, 38], whereas the reverse
situation, when CTCF-binding sequence does not block
promoter activation by enhancer, is not documented.
However, the existence of this kind of sequences is notunlikely since CTCF protein has multiple functions notnecessarily connected with blocking of enhancers (see[18] for review).
The extent of blocking of promoter activation byenhancer corrected for silencer activity of the fragment
varies from 2.4-fold (fragment C2 in plus orientation) toalmost 7-fold (fragment C8 in minus orientation). Besides,
there are fragments whose enhancer-blocking activity
depend on orientation relative to promoter (C8) and havepractically no dependence on the orientation (C3, C6).
It is interesting that fragment C3, which was not
identified as an insulator by its chromatin structure properties [35, 36], but able to strongly bind CTCF (Table 1),
also displayed high enhancer-blocking activity.
Using the CTCF binding site database (http://insulatordb.uthsc.edu/storm.php), we identified within five
selected DNA fragments the most probable 20 bp binding901

sites for CTCF. Fragment C6 contained two such sites(Fig. 2b). Using these sequences and the WebLogo server(http://weblogo.berkeley.edu/logo.cgi), we constructed
the consensus sequence logo for CTCF binding qualitatively coinciding with a similar consensus constructedbased on the whole genome data [39]. However, we foundno correlation between the number of nucleotides thatcoincide with the consensus and enhancer-blocking
activity of the DNA fragment. Thus, within sequence C8with the highest enhancer-blocking efficiency, four
nucleotides out of 20 was found to coincide with the consensus, whereas within sequence C4 with the lowest
blocking efficiency there were nine such nucleotides.
Most likely the enhancer-blocking efficiency is determined not only by CTCF binding, but also by other factors, e.g. by the ability to bind additional proteins.
Enhancer-blocking activity of cHS4 insulator fromchicken b-globin locus. The full-length (1.2 kb) cHS4
chicken β-globin insulator [3, 31] is a typical CTCFbinding element that displays enhancer-blocking activity
in different systems, including transient transfection assay
with constructions containing SV40 promoter andenhancer [26].
We inserted the cHS4 insulator in both orientations
both between SV40 enhancer and promoter and between

Fig. 4. Enhancer-blocking activity of potential insulators identified in the human genome by positive–negative selection with respect to SV40
promoter–enhancer pair. Data for cell lines HeLa and CHO are shown. The luciferase activity was normalized to that of pGL4EPV.

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promoter SV40 and enhancer E2 identified in this work(see above). In parallel, control constructs were preparedwith the cHS4 insulator inserted outside of the promoter–enhancer pair and constructs not containing insulator.
The plasmids were transfected into HeLa cells, andluciferase activity was measured (Fig. 3). As seen in Fig.
3a, the cHS4 insulator is able to reduce the activity of the
SV40 promoter–enhancer pair 2-3-fold only when located between these elements. The enhancer-blocking effectwas not observed when phage λ DNA fragment wascloned between promoter and enhancer. Similar results
were obtained for the SV40 promoter–E2 enhancer pair.
In both cases, the enhancer-blocking activity barely
depended on the orientation of the insulator relative tothe promoter (Fig. 3b).
Enhancer-blocking activity of fragments identified bypositive–negative selection. Four fragments identified earlier (see Table 2) were inserted into pGL4EPV2 plasmid
between SV40 promoter and enhancer. Since the selectionof the enhancer-blocking fragments was initially done in
CHO cells, the constructions were linearized and transfected in CHO and, for comparison, into HeLa cells. The
results are presented in Fig. 4. As seen, three out of four(75%) of the selected fragments significantly (2-3-fold)reduced the activity of the SV40 promoter–enhancer pair
independent of their orientation relative to the promoter(not shown). Moreover, the enhancer-blocking activity of
two elements (Ins2 and Ins3) was detected only in CHO
cells and was practically absent in HeLa, while the activity of Ins4 was identical in these cell lines. Notably, theenhancer-blocking activity of the CTCF-binding elements Ins2 and Ins3 (Table 2) was dependent on cell type,whereas the activity of Ins4, which does not bind CTCF,
was independent of cell type. However, more study is necessary to confirm this correlation.
The results obtained here show that the transienttransfection of cultured cells with linearized plasmids
allows revealing the enhancer-blocking activity of potential insulators including both the standard cHS4 chicken
β-globin insulator and several DNA fragments selectedfrom the human genome sequence. The activity of different sequences is characterized by certain tissue specificity and by dependence on orientation of the fragments relative to the promoter. Thus, the transfection model can beused for quantitative analysis of the enhancer-blocking
activity of potential insulators. The enhancer-blocking
activity of different sequences can be characterized by tissue-specificity and orientation-dependence relative tothe promoter. We conclude that the system used is inmany cases an adequate instrument for quantitative
analysis of enhancer-blocking activity.
The authors are grateful to Gaelle M. Lefevre andGary Felsenfeld (NIDDK, National Institutes of Health,
USA) for gift of pJC13-1 plasmid containing cHS4 insulator.

This work was supported by the Scientific School
program (project NSh 2395.2008.4), the Program of theRussian Academy of Sciences on Molecular and Cellular
Biology, and by the Russian Foundation for Basic
Research (project 10-04-01472).

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