New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity.

In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains.

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality.

Background: CTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer-promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries.

Boundary bypass activity in the region of the bithorax complex is position dependent and regulated.

Expression of () in abdominal segments A5A8 is controlled by four regulatory domains, . Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements (, , and ).

Transcriptional Readthrough Interrupts Boundary Function in Drosophila.

In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized bithorax complex (BX-C) to study one potential mechanism for controlling boundary function.

Boundary Bypass Activity in the Region of the Bithorax Complex is Position Dependent and Regulated.

Unlabelled: Expression of ( ) in abdominal segments A5 A8 is controlled by four regulatory domains, . Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements ( , , and ).

Transcriptional read through interrupts boundary function in Drosophila.

Unlabelled: In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. In the studies reported here we have taken advantage of the well characterized Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization.

Mechanisms of Interaction between Enhancers and Promoters in Three Model Systems.

In higher eukaryotes, the regulation of developmental gene expression is determined by enhancers, which are often located at a large distance from the promoters they regulate. Therefore, the architecture of chromosomes and the mechanisms that determine the functional interaction between enhancers and promoters are of decisive importance in the development of organisms. Mammals and the model animal have homologous key architectural proteins and similar mechanisms in the organization of chromosome architecture.

Mechanisms of enhancer-promoter communication and chromosomal architecture in mammals and .

The spatial organization of chromosomes is involved in regulating the majority of intranuclear processes in higher eukaryotes, including gene expression. Drosophila was used as a model to discover many transcription factors whose homologs play a key role in regulation of gene expression in mammals. According to modern views, a cohesin complex mostly determines the architecture of mammalian chromosomes by forming chromatin loops on anchors created by the CTCF DNA-binding architectural protein.

The Variable CTCF Site from Drosophila melanogaster Ubx Gene is Redundant and Has no Insulator Activity.

CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the surrounding chromatin.

Fragments of the Fab-3 and Fab-4 Boundaries of the Drosophila melanogaster Bithorax Complex That Include CTCF Sites Are not Effective Insulators.

The segment-specific regulatory domains of the Bithorax complex (BX-C), which consists of three homeotic genes Ubx, abd-A and Abd-B, are separated by boundaries that function as insulators. Most of the boundaries contain binding sites for the architectural protein CTCF, which is conserved for higher eukaryotes. As was shown previously, the CTCF sites determine the insulator activity of the boundaries of the Abd-B regulatory region.

The Piragua Gene Is not Essential for Drosophila Development.

Proteins with clusters of CH zinc finger domains (CH-proteins) constitute the most abundant class of transcription factors in higher eukaryotes. N-terminal ZAD (zinc finger-associated domain) dimerization domain has been identified in a large group of CH-proteins mostly in insects. The piragua gene encodes one of these proteins, Fu2.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster.

Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein-protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain.

Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila.

The Abdominal-B (Abd-B) gene belongs to the bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold.

Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190.

CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes.

Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster.

Background: Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them.

Mapping of functional elements of the Fab-6 boundary involved in the regulation of the Abd-B hox gene in Drosophila melanogaster.

The autonomy of segment-specific regulatory domains in the Bithorax complex is conferred by boundary elements and associated Polycomb response elements (PREs). The Fab-6 boundary is located at the junction of the iab-5 and iab-6 domains. Previous studies mapped it to a nuclease hypersensitive region 1 (HS1), while the iab-6 PRE was mapped to a second hypersensitive region HS2 nearly 3 kb away.

Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes.

In higher eukaryotes, enhancers determine the activation of developmental gene transcription in specific cell types and stages of embryogenesis. Enhancers transform the signals produced by various transcription factors within a given cell, activating the transcription of the targeted genes. Often, developmental genes can be associated with dozens of enhancers, some of which are located at large distances from the promoters that they regulate.

The insulator functions of the polydactyl C2H2 zinc finger protein CTCF: Necessity versus sufficiency.

In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). Here, we have determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw).

Small Drosophila zinc finger C2H2 protein with an N-terminal zinc finger-associated domain demonstrates the architecture functions.

Recently, the concept has arisen that a special class of architectural proteins exists, which are responsible not only for global chromosome architecture but also for the local regulation of enhancer-promoter interactions. Here, we describe a new architectural protein, with a total size of only 375 aa, which contains an N-terminal zinc finger-associated domain (ZAD) and a cluster of five zinc finger C2H2 domains at the C-terminus. This new protein, named ZAD and Architectural Function 1 protein (ZAF1 protein), is weakly and ubiquitously expressed, with the highest expression levels observed in oocytes and embryos.

Distinct Elements Confer the Blocking and Bypass Functions of the Bithorax Boundary.

Boundaries in the bithorax complex (BX-C) enable the regulatory domains that drive parasegment-specific expression of the three genes to function autonomously. The four regulatory domains (, , , and ) that control the expression of the () gene are located downstream of the transcription unit, and are delimited by the , , , and boundaries. These boundaries function to block cross talk between neighboring regulatory domains.

Study of dCTCF Insulator Activity in Drosophilamelanogaster Model Systems.

Using transgenic Drosophila model systems, we showed that four binding sites for the architectural protein dCTCF per se cannot form an effective insulator that blocks enhancers and protects against the Polycomb-dependent repression. These results suggest that, in the known Drosophila insulators, the dCTCF protein functions in cooperation with other architectural proteins.

Complete reconstitution of bypass and blocking functions in a minimal artificial insulator from complex.

Boundaries in the complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the genes , and The boundary is located between the and domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between and the promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the and domains; however, they lack bypass activity, and is unable to regulate Here we show that an ∼200-bp sequence of dHS1 from the boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF.

Boundaries mediate long-distance interactions between enhancers and promoters in the Drosophila Bithorax complex.

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously.

The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7.