Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function

Abstract

Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

“…The ChIP-seq together with the LC-MS/MS results of the HIPP1-BioTAP pull-down suggest that the protein associates with HP1a within heterochromatin and also plays an HP1a-indpendent role within insulator complexes in euchromatin. The association with insulators is further strengthened by the recent identification of CG8436 and CG9740 (Table 3) as novel CP190-interacting proteins in S2 cells (Cuartero et al 2014). Overall, our analyses of HIPP1 suggest separate roles in the euchromatic and heterochromatic portions of the genome, but we cannot exclude the possibility that the presence of HIPP1 simultaneously in both locations has some common purpose.…”

Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

“…This argues that the cobinding of Su(Hw), CTCF, Cp190, and Mod(mdg4) or other recently identified Cp190-interacting proteins (Pita, Ibf1, Ibf2, and ZIPIC) (Cuartero et al 2014;Maksimenko et al 2015) does not automatically predict a strong enhancer-blocking ability. It appears that Fub has additional features that make it an exceptionally strong insulator element.…”

“…Of all Drosophila insulator proteins, only CTCF has a clear ortholog in mammals (Baniahmad et al 1990;Lobanenkov et al 1990). Multiple lines of evidence indicate that insulator proteins act as multisubunit complexes (Matzat and Lei 2014).In addition, genomic mapping shows that insulator proteins bind chromatin in distinct combinations (Negre et al 2010;Schwartz et al 2012;Cuartero et al 2014;Maksimenko et al 2015). Importantly, only certain combinations of insulator proteins make these elements capable of blocking enhancerpromoter communications, suggesting that these proteins have additional unrelated functions.…”

“…Based on their biochemical and functional properties, the known Drosophila insulator proteins can be divided in three groups. The first group consists of nine sequence-specific DNA binding proteins: Su(Hw), CTCF, BEAF-32, Ibf1, Ibf2, Pita, ZIPIC (also known as CG7928), Dwg (also known as Zw5), and GAF (the product of Trithorax-like gene) (Geyer and Corces 1992;Zhao et al 1995;Gaszner et al 1999;Schweinsberg et al 2004; Copyright © 2016 Moon Cuartero et al 2014;Maksimenko et al 2015;Wolle et al 2015). The second group includes Cp190 and multiple protein isoforms encoded by the mod(mdg4) gene (Dorn et al 2001;Pai et al 2004;.…”

“…In addition, genomic mapping shows that insulator proteins bind chromatin in distinct combinations (Negre et al 2010;Schwartz et al 2012;Cuartero et al 2014;Maksimenko et al 2015). Importantly, only certain combinations of insulator proteins make these elements capable of blocking enhancerpromoter communications, suggesting that these proteins have additional unrelated functions.…”