Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.

According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer–promoter interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. Here we have shown that the ZAD domains of ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene promoter. However, no functional interaction between binding sites for different proteins has been revealed, suggesting that such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.

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Boundaries in the Bithorax complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including Fab-7 and Mcp. To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6↔iab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7, we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7: it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.

A special class of poorly characterized architectural proteins is required for chromatin topology and enhancer–promoter interactions. Here, we identify Opbp as a new Drosophila architectural protein, interacting with CP190 both in vivo and in vitro. Opbp binds to a very restrictive set of genomic regions, through a rare sequence specific motif. These sites are co-bound by CP190 in vivo, and generally located at bidirectional promoters of ribosomal protein genes. We show that Opbp is essential for viability, and loss of opbp function, or destruction of its motif, leads to reduced ribosomal protein gene expression, indicating a functional role in promoter activation. As characteristic of architectural/insulator proteins, the Opbp motif is sufficient for distance-dependent reporter gene activation and enhancer-blocking activity, suggesting an Opbp-mediated enhancer–promoter interaction. Rather than having a constitutive role, Opbp represents a new type of architectural protein with a very restricted, yet essential, function in regulation of housekeeping gene expression.