Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

Abstract

Enhancer-mediated promoter activation is a fundamental mechanism of gene regulation in eukaryotes (10, 44). Recently, sequences in different organisms have been identified that constrain enhancer action. These elements, known as insulators, block communication between an enhancer and promoter only when the insulator is positioned between these regulatory elements. Similarly, insulators prevent silencer interactions with promoters (6,10,30,44,45). The properties of insulators are exemplified by the gypsy insulator that originally was found in the gypsy transposable element (19,24).Genetic and molecular approaches have led to the identification and characterization of three proteins, Suppressor of Hairy wing [Su(Hw)], Mod(mdg4)-67.2, and CP190, that are required for the activity of the gypsy insulator (6, 36). Su(Hw) is a zinc finger protein that binds 12 directly repeated copies of a short sequence motif in the gypsy insulator (9, 42). In addition, Su(Hw) has two acidic domains located at the amino (N) and carboxyl (C) termini of the protein and a C-terminal enhancer-blocking region that is essential for insulation (23, 29). The mod(mdg4) gene, also known as E(var)3-93D, encodes a large set of protein isoforms with specific functions in regulating the chromatin structure of different genes (3). All isoforms encoded by mod(mdg4) contain a BTB/POZ domain and a glutamine-rich (Q) region in the N terminus (3, 7). The BTB (broad complex, tramtrack, bric-a-brac) or POZ (poxvirus and zinc finger) domain identifies a large family of proteins in organisms from yeast to humans (43,47). This domain functions as a protein interaction domain that facilitates homodimer (2, 33, 34) and heterodimer formation as well as oligomerization (11,28,37). One of the mod(mdg4)-encoded protein isoforms, Mod(mdg4)-67.2, interacts with the enhancer-blocking domain of the Su(Hw) protein (12, 20) through a C-terminal acidic domain. This domain is affected in two viable mutations mod(mdg4) u1 and mod(mdg4) T6 (12, 15). The third component of the insulator complex, CP190, also contains a BTB domain (38). It was suggested that Mod(mdg4)-67.2 and CP190 interact through their BTB domains.The mod(mdg4) u1 and mod(mdg4) T6 mutations have varying effects on insulator function, resulting in partial restoration of enhancer-promoter communication in some cases, while transforming the insulator into a silencer in others (4,12,13,14,15,22). The domains of the Mod(mdg4)-67.2 protein required for the insulator and antirepression activity are not determined. Although the essential role of the BTB domain for Mod-(mdg4)-67.2 activity was predicted in the previous studies, this postulate has not been proven (12,20). Here we examined the role of the BTB domain in the functional activities of the Mod(mdg4)-67.2 protein.The structure of the BTB domain has been examined for mammalian transcriptional repressors 34). The high degree of sequence identity between the BTB domains of Mod(mdg4), Bcl-6, and PZLF (1) allowed us to predict key residues of the Mod(mdg4)-67.2 ...

SUMO conjugation is required for the assembly ofDrosophilaSu(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation

“…Although analysis of the CP190 sequence suggests the presence of two SIMs, we have not observed a direct interaction between CP190 and SUMO in vitro. However, CP190 and Mod(mdg4)-67.2 contain several protein-protein interaction domains, including BTB/POZ Pai et al, 2004), that might be involved in direct interaction with many DNA-binding transcription factors, such as Su(Hw) and dCTCF, to facilitate their assembly into the insulator bodies (Gerasimova et al, 2007;Golovnin et al, 2007;Mohan et al, 2007;Pai et al, 2004). It is noteworthy that heat shock has proved to induce redistribution of CP190 to the nuclear periphery, in complex with SUMO.…”

“…It was shown previously that Mod(mdg4)-67.2 facilitates Su(Hw) binding to insulator sequences (Gerasimova et al, 2000;Golovnin et al, 2007). The in vivo binding of the Su(Hw) and Mod(mdg4)-67.2 proteins to the gypsy and four endogenous Su(Hw) insulators (Parnell et al, 2006) was examined by ChIP analysis of chromatin isolated from the middle pupa stage (Fig.…”

“…Mod(mdg4)-67.2 is one of isoforms produced by the mod(mdg4) gene, also known as E(var)3-93D, which encodes a large set of proteins containing the BTB domain at the N-terminus and different C-terminal domains (Büchner et al, 2000). Mod(mdg4)-67.2 interacts with the enhancer-blocking domain of the Su(Hw) protein through an isoform-specific C-terminal acidic domain (Gause et al, 2001;Ghosh et al, 2001;Golovnin et al, 2007). This domain is affected in two viable mutations, mod(mdg4) u1 and mod(mdg4) T6 .…”

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

“…Although both Cp190 and Mod(mdg4) have BTB/POZ domains, these domains differ in their amino acid sequences and their protein-protein interaction properties. Thus in vitro and in yeast two-hybrid assays, the BTB/POZ domains of Cp190 form homodimers (Bonchuk et al 2011;Vogelmann et al 2014) while the BTB/POZ domains of Mod(mdg4) form homo-and heterotypic multimers with the BTB/POZ domains of several other members of the tramtrack group (Golovnin et al 2007;Bonchuk et al 2011). Furthermore, the isolated BTB/POZ domains of Cp190 and Mod(mdg4) do not interact with each other (Bonchuk et al 2011).…”

Insulator and Ovo Proteins Determine the Frequency and Specificity of Insertion of the gypsy Retrotransposon in Drosophila melanogaster

“…The molecular basis for interactions between individual insulators is not well understood but it has been suggested that Modifier of mdg4 [Mod(mdg4)] and CP190, both components of the insulator complex, might facilitate such interactions by mediating proteinprotein contacts between the BTB domains present in the two proteins (Gause et al 2001;Ghosh et al 2001;Golovnin et al 2007). These two properties of gypsy, sitespecific integration and insulator activity, have been the subject of intense but unrelated studies during the past two decades (Mevel-Ninio et al 1989;Bucheton 1995;Prud'homme et al 1995;Dej et al 1998;Gerasimova and Corces 2001;Labrador and Corces 2001;Capelson and Corces 2004).…”