Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements

Abstract

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs–scs, scs’–scs’, 1A2–1A2 and Wari–Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5–Su(Hw), dCTCF–Su(Hw), or dCTCF–Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

Two new insulator proteins, Pita and ZIPIC, target CP190 to chromatin

“…6B,D). Similar results had been previously obtained using binding sites for the known insulator proteins Su(Hw) and Dwg (also known as Zw5) (Gaszner et al 1999;Scott et al 1999;Golovnin et al 2003;Kyrchanova et al 2008aKyrchanova et al , 2013. Thus, Pita and ZIPIC function like insulator proteins by blocking enhancers and protecting the reporter gene expression from PRE-mediated silencing.…”

Drosophila BTB/POZ Domains of “ttk Group” Can Form Multimers and Selectively Interact with Each Other

“…Hence, we decided to test the ability of GAF to support the enhancerpromoter communication in Drosophila using the GAL4/white assay described previously. 35,36 This assay is based on the finding that the yeast GAL4 activator bound to sites located upstream of the yellow gene fails to stimulate the white promoter placed downstream of the yellow 3′ end. The DNA fragment containing five binding sites for GAF was constructed as described previously.…”

Insulators form gene loops by interacting with promoters in Drosophila

“…X63731), the 858 bp sequence corresponding to the Fab-7 insulator (PCR amplified with primers 5Ј-GATTTCAAGCTGTGTGGCGGGGG-3Ј and 5Ј-CGTGAGCGACCG -AAACTCG-3Ј), the 350 bp Mcp insulator sequence (PCR amplified with primers 5Ј-GCTCAGAGTACATAAGCG-3Ј and 5Ј-CCCAATCGTTGT -AAGTGT-3Ј) and CTCF ϫ4 were obtained as described (Kyrchanova et al, 2008a;Kyrchanova et al, 2008b). Sequences corresponding to the scs, Fab-7, Mcp insulators and CTCF ϫ4 fragment were cloned into pBluescript SK+ between lox sites to produce lox(scs), lox(Fab-7 R ), lox(MCP R ) and lox(CTCF ϫ4R ) plasmids, respectively.…”

“…Experiments with transgenic lines suggest that the interaction between insulators can result in the formation of chromatin loops that either block or facilitate long-distance enhancer-promoter communication depending on the nature of the interacting insulators as well as on the distances between all the elements involved (enhancers, insulators and promoters) and their relative 'strength' (Muravyova et al, 2001;Cai and Shen, 2001;Conte et al, 2002;Gruzdeva et al, 2005;Savitskaya et al, 2006;Kyrchanova et al, 2008a). Alternatively, insulator action can be explained by the ability of insulators to form direct contacts with either an enhancer (the decoy model) or a promoter, thereby inactivating them.…”