Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus

Abstract

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. We developed an experimental strategy to identify silencers and EBs using transient transfection assays. A known insulator and EB from the chicken beta-globin locus, cHS4, served as a control element for these assays. We examined 47 sequences from a 1.8-Mb region of human chromosome 7 for silencer and EB activities. The majority of functional elements displayed directional and promoter-specific activities. A limited number of sequences acted in a dual manner, as both silencers and EBs. We examined genomic data, epigenetic modifications, and sequence motifs within these regions. Strong silencer elements contained a novel CT-rich motif, often in multiple copies. Deletion of the motif from three regions caused a measurable loss of silencing ability in these sequences. Moreover, five duplicate occurrences of this motif were identified in the cHS4 insulator. These motifs provided an explanation for an uncharacterized silencing activity we measured in the insulator element. Overall, we identified 15 novel NREs, which contribute new insights into the prevalence and composition of sequences that negatively regulate gene expression.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…20,38 We therefore designed a two-reporter gene assay implementing the accuracy of the former assay, but in which polar insulating activities can be distinguished from enhancer inhibition, global gene silencing effects or gene copy number variations (Figure 1a, construct-2). Changes in the expression of the reporter and reference genes were assessed by cytofluorometry from the green fluorescent protein (GFP) and blue fluorescent protein (BFP) fluorescence profiles of single cells within populations of transiently transfected HeLa cells.…”

Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif

“…DNA segments that cause a significant increase in expression of luciferase are enhancers. This assay is a well-recognized approach to analyzing the function of CRMs, having been used in experiments ranging from the early definition of enhancers (Banerji et al 1981;Mellon et al 1981) to many experiments screening noncoding segments of genomic DNA for effects on expression (e.g., Frazer et al 2004;Baroukh et al 2005;Grice et al 2005;Wang et al 2006;Petrykowska et al 2008). This assay provided a tractable method to test a biological activity of the 123 DNA segments examined in this study.…”

Conserved and non-conserved enhancers direct tissue specific transcription in ancient germ layer specific developmental control genes

“…Also the possibility exists that these CNEs could be negative regulatory elements and hence our assay fails to capture this activity. Some recent results from other groups have indicated the same for the human genome [15]. Also other functions such as chromatin attachment sites, miRNA genes or splice regulatory regions may also reside in such highly conserved non-coding sequences [50-54].…”