The Phylogenetic Distribution of Non-CTCF Insulator Proteins Is Limited to Insects and Reveals that BEAF-32 Is Drosophila Lineage Specific

Abstract

Chromatin insulators are DNA sequences found in eukaryotes that may organize genomes into chromatin domains by blocking enhancer-promoter interactions and preventing heterochromatin spreading. Considering that insulators play important roles in organizing higher order chromatin structure and modulating gene expression, very little is known about their phylogenetic distribution. To date, six insulators and their associated proteins have been characterized, including Su(Hw), Zw5, CTCF, GAF, Mod(mdg4), and BEAF-32. However, all insulator proteins, with the exception of CTCF, which has also been identified in vertebrates and worms, have been exclusively described in Drosophila melanogaster. In this work, we have performed database searches utilizing each D. melanogaster insulator protein as a query to find orthologs in other organisms, revealing that except for CTCF all known insulator proteins are restricted to insects. In particular, the boundary element-associated factor of 32 kDa (BEAF-32), which binds to thousands of sites throughout the genome, was only found in the Drosophila lineage. Accordingly, we also found a significant bias of BEAF-32 binding sites in relation to transcription start sites (TSSs) in D. melanogaster but not in Anopheles gambiae, Apis mellifera, or Tribolium castaneum. These data suggest that DNA binding proteins such as BEAF-32 may have a dramatic impact in the genome of single evolutionary lineages. A more thorough evaluation of the phylogenetic distribution of insulator proteins will allow for a better understanding of whether the mechanism by which these proteins exert their function is conserved across phyla and their impact in genome evolution.

Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi

“…This similarity extends to the ability to modulate gene expression both positively and negatively and the imposition of polar effects on transcriptional units near but not flanked by the insulator DNA (41). This similarity also supports the hypothesis that the functional requirements for these insulator-like elements may have been conserved -2 G-3 G-4 G-5 G-6 G-7 G-8 G-9 G-10 G-11 G-12 G-13 G-14 G-15 G-16 G-17 G-18 evolutionarily over the >250 million years that these taxa are estimated to have diverged (45,46). Although the DNA components of the insulating elements were found first associated with the gypsy retrotransposon, it was shown later that there are independent gypsy-like and functional endogenous sequences in the D. melanogaster genome (47).…”

The BEAF-32 insulator coordinates genome organization and function during the evolution of Drosophila species

“…For these pairs, it is reasonable to predict the existence of regulatory mechanisms that functionally separate the two genes in order to attain the observed differential transcription. BEAF-32 is restricted to the Drosophila species (Schoborg and Labrador 2010), and mammalian cells may use other insulator proteins to accomplish this goal. In Drosophila there are several types of insulator elements that show different genomic distributions with respect to genes (Bushey et al 2009;Nègre et al 2010).…”

“…The distribution of the dCTCF insulator partially overlaps that of BEAF-32. Since CTCF is conserved between Drosophila and humans (Moon et al 2005;Schoborg and Labrador 2010), it is possible that this protein functionally replaces BEAF-32 in maintaining differential transcription programs in genes located in close head-tohead gene pairs. When the human genome was specifically examined for the organization of close head-to-head gene pairs, those containing CTCF showed lower correlation of expression, suggesting that this mechanism may be also conserved in humans (Xie et al 2007).…”

“…Insulators are good candidates to perform such function given their ability to regulate enhancer-promoter interactions. More specifically, the BEAF-32 insulator protein is highly conserved in Drosophila and its presence appears to be restricted to this genus (Schoborg and Labrador 2010). We therefore examined the genome-wide distribution of BEAF-32 in D. melanogaster embryos using ChIPseq, and found BEAF-32 frequently located between close adjacent genes oriented head-to-head (Fig.…”

The BEAF-32 insulator protein is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes

“…Non-CTCF insulators appear to be restricted to arthropods, and among several insect species examined (Anopheles gambiae, Apis mellifera or Tribolium castaneum), BEAF was present exclusively in the Drosophila genus (Heger et al, 2013;Schoborg and Labrador, 2010). We speculate that conserved signaling modules of the Hippo pathway in growth control may be co-opted for cell fate specification by regulatory factors such as BEAF that are unique to dipterans.…”