Description

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.

Methods

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.

Credits

These data were produced by the Elnitski lab at NHGRI, NIH. (contact: elnitski@mail.nih.gov)

References

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.

Data Release Policy

Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until column on the track configuration page and the download page. The full data release policy for ENCODE is available here.