Interaction between a pair of gypsy insulators or between heterologous gypsy and Wari insulators modulates Flp site-specific recombination in Drosophila melanogaster

Abstract

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. An Flp technology was used to examine interactions between Drosophila gypsy and Wari insulators in somatic and germ cells. The gypsy insulator consists of 12 binding sites for the Su(Hw) protein, while the endogenous Wari insulator, located on the 3' side of the white gene, is independent from the Su(Hw) protein. Insertion of the gypsy but not Wari insulator between FRT sites strongly blocks recombination between Flp dimers bound to FRT sites located on the same chromatid (recombination in cis) or in sister chromatids (unequal recombination in trans). At the same time, the interaction between Wari and gypsy insulators regulates the efficiency of Flp-mediated recombination. Thus, insulators may have a role in controlling interactions between distantly located protein complexes (not only those involved in transcriptional gene regulation) on the same chromosome or on sister chromatids in somatic and germ cells. We have also found that the frequency of Flp-mediated recombination between FRT sites is strongly dependent on the relative orientation of gypsy insulators. Taken together, our results indicate that the interactions between insulators can be visualized by Flp technology and that insulators may be involved in blocking undesirable interactions between proteins at the two-chromatid phase of the cell cycle.

Mechanism of Chromosomal Boundary Action: Roadblock, Sink, or Loop?

“…Similarly, in vitro experiments have shown that the formation of a looped domain that topologically isolates the NtrCdependent enhancer from the Escherichia coli glnAp2 promoter in a closed supercoiled plasmid is sufficient to disrupt transcriptional activation (Bondarenko et al 2003). Further support for the idea that boundaries function by subdividing the chromosome into topologically independent looped domains comes from studies on how insulators affect FLP-mediated recombination (Krivega et al 2010). These authors found that placing a single su(Hw) boundary in between two FRT sites substantially suppresses FLP-mediated recombination.…”

Effective Blocking of the White Enhancer Requires Cooperation between Two Main Mechanisms Suggested for the Insulator Function

“…Previously, we have used the Flp-recombination assay [53] to demonstrate that the pairing between gypsy insulators strongly depends on their relative orientation. According to our model, the orientation-dependent effect is explained by the involvement of at least two insulator-bound proteins in specific protein-protein interactions.…”

“…The BTB domain is located at the N-terminus of Mod(mdg4)-67.2 and mediates homo-multimerization [52]. There are many evidences of functional distant interactions between the gypsy insulators [9], [11], [53], which were recently confirmed by the 3C method [54].…”

tDNA insulators and the emerging role of TFIIIC in genome organization

“…10,[45][46][47][48] In each case, DNA-bound insulator proteins require the recruitment of additional proteins for insulator function, suggesting active insulator complexes are regulated both by the recruitment of insulator proteins to DNA, and the recruitment of essential co-factors. 49 Mammalian CTCF specifically recruits the cohesin complex, 50,51 a ring-shaped structure that mediates cohesion between sister chromatids from S-phase until mitosis, which may stabilize CTCF-mediated physical interactions through a similar mechanism.…”

“…6,9,58 Meanwhile, insulator proteins are distributed across the genome at thousands of sites, 32,[59][60][61][62][63] and are significantly enriched at the borders of laminaassociated domains, 64,65 consistent with microscopy-based staining of perinuclear insulator bodies. Numerous studies have also characterized the ability of CTCF and Drosophila insulator proteins to mediate locus specific interactions, [45][46][47][48] and recent genome-wide profiling of CTCF interactions in mouse ES cells suggests CTCF facilitates coregulation of related genes by establishing chromatin loops enriched for active or repressive epigenetic signatures, and by bridging interactions between enhancers and promoters. chromosomal interactions (Fig.…”