Silencers and enhancer-blockers (EBs) are cis-acting, negative regulatory
elements (NREs) that control interactions between promoters and enhancers.
Although relatively uncharacterized in terms of biological mechanisms, these
elements are likely to be abundant in the genome. Examples of NREs include
silencers, which decrease expression of a gene under their regulation and
enhancer-blocking (EB) elements, which prevent the action of an enhancer on
a promoter when placed between the two, but not otherwise. Examples of NREs
are extremely limited. EB and silencer assays typically require integration
of the reporter gene into the chromatin of genomic DNA. The process is too
laborious to use in large-scale analyses. To improve scalability, the system
uses non-integrated recombinant plasmids, which have been shown to support
EB activity. Silencing and EB function in genomic sequences were evaluated
by developing a transient transfection assay suitable for high-throughput
screening. To ensure that the identified elements are capable of overcoming the
effects of strong enhancers, the assay utilizes an enhancer from the human
beta-globin locus control region. Known as DNase I hypersensitive site II
(HS2) this enhancer is functional in multiple cell lines, with multiple
promoters. The presence of HS2 provides a large window of expression to
reliably measure loss-of-function effects.
This track was developed as part of the ENCODE project. It currently is
limited to chromosome 7.
To assess transient
expression, 4 x 105 K562 cells and 6 x 104 HeLa or 293T cells were transfected
with 0.4 micrograms of test DNA and 4.0 or 40 nanograms of pRL-Tk Renilla
plasmid for lipofection (using the reagent TFX-50) or electroporation (using
the Amaxa 96-well nucleofector II). Both approaches used Renilla luciferase as
a control for transfection efficiency. Luciferase expression was measured in a
96-well plate format with detection of fluorescence using the dual luciferase
"Stop and Glo" procedure from Promega. Measurements were recorded on a Berthold
plate-reader luminometer. The average expression level from three replicate
transfections was normalized to the Renilla luciferase co-transfection
control. This value was further normalized to the average expression level
from three normalized replicates of the promoter-only plasmid to yield a "fold"
enhancement measurement. Upon producing a silencing phenotype, each construct
was re-sequenced to confirm the integrity of the plasmid.
These data were produced by the Elnitski lab
at NHGRI, NIH. (contact:
Petrykowska HM, Vockley CM, Elnitski L.
Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus.
Genome Res. 2008 Aug;18(8):1238-46.
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