ISSN 10227954, Russian Journal of Genetics, 2010, Vol. 46, No. 1, pp. 13–19. © Pleiades Publishing, Inc., 2010.
Original Russian Text © M.M. Erokhin, P.G. Georgiev, D.A. Chetverina, 2010, published in Genetika, 2010, Vol. 46, No. 1, pp. 18–25.
The presentday data demonstrate that enhancers
can stimulate promoters covering distances of up to
tens of thousands of base pairs . It is supposed that
the main role of insulators is to ensure specificity of
interactions between enhancers and promoters in the
process of development .
There are two basic criteria for a regulatory element
to be considered an insulator . First, insulators
block the activity of enhancers only in case an insula
tor is placed between an enhancer and a promoter.
Second, insulators can act as a barrier between the
active chromatin and the condensed chromatin and
promote the independent of the surrounding chroma
tin expression of a transgene integrated into the
Despite numerous models proposed for the action
of insulators, the mechanisms of their functioning
remain unclear [2, 4].
According to structural models of the action of
insulators, their ability to block enhancers is a result of
the organization by insulators of a highorder chroma
tin structure [3, 4]. It is postulated that physical
attachment of proteins of the insulator complex either
to the nuclear matrix or to the nuclear envelope leads
to the formation of loops that are independent
domains of gene transcription. In this case, the
enhancer located in one chromatin domain is not able
to activate the promoter from the other domain. How
ever, this group of models has not received a reliable
experimental support. Moreover, as shown in previous
studies, the functional interactions between
insulators can stimulate both insulation and acti
vation of promoters by enhancers [5, 10].
Many conclusions were drawn from studying the
interaction between two copies of the same insulator.
Additional studies of functional interactions between
different insulators are needed for a more complete
understanding of an actually possible effect of interac
tions between insulators in the genome.
Su(Hw) is the best studied insulator in
It was discovered in the 5'untranslated region of the
) retrotransposon [11, 12]. The DNA
sequence of the Su(Hw) insulator contains 12 binding
sites for the Su(Hw) (Suppressor of Hairy wing) pro
tein [13, 14]. In addition to the Su(Hw) protein, the
enhancerblocking activity of this insulator requires
the proteins Mod(mdg4)67.2 (modifier of mdg4) and
CP190 (centrosomal protein 190) [15, 18].
The Wari insulator was found in the immediate
vicinity of the
gene end . The activity of Wari
is determined by new, as yet unidentified, transcription
factors, which suggests a far greater diversity of insula
tor proteins than thought earlier. The Wari insulator
showed itself able to functionally interact with heterol
ogous Su(Hw)dependent insulators .
In this work, we continued studying the functional
interactions between two nonhomologous insulators,
Su(Hw) and Wari, and the role of a loop formed as a
result of their interaction in the creation of indepen
dent domains of gene expression.
MATERIALS AND METHODS
Development of plasmid constructs.
containing a 825bp Wari insulator (
ident insulator), and plasmid vectors containing a Wari
insulator between lox and frt sites, were obtained ear
Effects of Functional Interactions between Nonhomologous
Insulators Wari and Su(Hw)
M. M. Erokhin, P. G. Georgiev, and D. A. Chetverina
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia;
Received May 21, 2009
—Insulators are regulatory DNA elements restricting gene activation by enhancers. Interactions
between insulators can lead to both insulation and activation of promoters by enhancers. In this work, we ana
lyzed the effects of interaction of two
insulators, Wari and Su(Hw). The functional interaction
between these insulators was found to enhance the activity of the Su(Hw) insulator only, but not of the Wari
insulator. This suggests that the formation of a chromatin loop between interacting insulators is not a key fac
tor for enhancement of insulation, which is in disagreement with the main idea of structural models. In addi
tion, the effect of interaction between Wari and Su(Hw) depends on a distance between them and on the posi
tion in the system relative to other regulatory elements.