The emergence of whole-genome assays has initiated numerous genome-wide studies of transcription factor localizations at genomic regulatory elements (enhancers, promoters, silencers, and insulators), as well as facilitated the uncovering of some of the key principles of chromosomal organization. However, the proteins involved in the formation and maintenance of the chromosomal architecture and the organization of regulatory domains remain insufficiently studied. This review attempts to collate the available data on the abundant but still poorly understood family of proteins with clusters of the C2H2 zinc finger domains. One of the best known proteins of this family is a well conserved protein known as CTCF, which plays a key role in the establishment of the chromosomal architecture in vertebrates. The distinctive features of C2H2 zinc finger proteins include strong and specific binding to a long and unique DNA recognition target sequence and rapid expansion within various animal taxa during evolution. The reviewed data support a proposed model according to which many of the C2H2 proteins have functions that are similar to those of the CTCF in the organization of the chromatin architecture.

BackgroundInsulators play a central role in gene regulation, chromosomal architecture and genome function in higher eukaryotes. To learn more about how insulators carry out their diverse functions, we have begun an analysis of the Drosophila CTCF (dCTCF). CTCF is one of the few insulator proteins known to be conserved from flies to man.ResultsIn the studies reported here we have focused on the identification and characterization of two dCTCF protein interaction modules. The first mediates dCTCF multimerization, while the second mediates dCTCF–CP190 interactions. The multimerization domain maps in the N-terminus of the dCTCF protein and likely mediates the formation of tetrameric complexes. The CP190 interaction module encompasses a sequence ~200 amino acids long that spans the C-terminal and mediates interactions with the N-terminal BTB domain of the CP190 protein. Transgene rescue experiments showed that a dCTCF protein lacking sequences critical for CP190 interactions was almost as effective as wild type in rescuing the phenotypic effects of a dCTCF null allele. The mutation did, however, affect CP190 recruitment to specific Drosophila insulator elements and had a modest effect on dCTCF chromatin association. A protein lacking the N-terminal dCTCF multimerization domain incompletely rescued the zygotic and maternal effect lethality of the null and did not rescue the defects in Abd-B regulation evident in surviving adult dCTCF mutant flies. Finally, we show that elimination of maternally contributed dCTCF at the onset of embryogenesis has quite different effects on development and Abd-B regulation than is observed when the homozygous mutant animals develop in the presence of maternally derived dCTCF activity.ConclusionsOur results indicate that dCTCF–CP190 interactions are less critical for the in vivo functions of the dCTCF protein than the N-terminal dCTCF–dCTCF interaction domain. We also show that the phenotypic consequences of dCTCF mutations differ depending upon when and how dCTCF activity is lost.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0168-7) contains supplementary material, which is available to authorized users.

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

The parasegment-specific expression of the three Drosophila Bithorax complex homeotic genes is orchestrated by nine functionally autonomous regulatory domains. Functional autonomy depends upon special elements called boundaries or insulators that are located between each domain. The boundaries ensure the independent activity of each domain by blocking adventitious interactions with initiators, enhancers and silencers in the neighboring domains. However, this blocking activity poses a regulatory paradox--the Bithorax boundaries are also able to insulate promoters from regulatory interactions with enhancers and silencers and six of the nine Bithorax regulatory domains are separated from their target genes by at least one boundary element. Here we consider several mechanisms that have been suggested for how the Bithorax regulatory domains are able to bypass intervening boundary elements and direct the appropriate parasegment-specific temporal and spatial expression of their target gene.