Abstract
Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.
Highlights
Mammalian gene expression is regulated in cis by DNA sequences that serve as binding sites for proteins which support or suppress transcription either by directly interacting with transcription factors or by modifying the surrounding chromatin
They include silencer elements derived from humans, rats, chickens, and yeast, with sizes ranging from 31 bp to 985 bp
The activities of some of these elements were assessed using the promoters and topological arrangements derived from their native loci, making it difficult to distinguish between context-dependent negative regulatory elements and context-independent autonomous silencers
Summary
Mammalian gene expression is regulated in cis by DNA sequences that serve as binding sites for proteins which support or suppress transcription either by directly interacting with transcription factors or by modifying the surrounding chromatin. Autonomous silencers function by establishing a repressive chromatin state that can be stably inherited [7] This is typically accomplished by recruiting proteins capable of modifying DNA (e.g. methyltransferases) or histone tails (e.g. histone deacetylases) in a manner that supports the formation of heterochromatin, or proteins that help stabilize and propagate heterochromatin (e.g. polycomb group proteins, heterochromatin protein 1). This in turn prevents activators and transcription factors from accessing gene promoters
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