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. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.

Boundary bypass activity in the abdominal-B region of the Drosophila bithorax complex is position dependent and regulated.

Expression of Abdominal-B (Abd-B) in abdominal segments A5-A8 is controlled by four regulatory domains, iab-5-iab-8. 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 (Mcp, Fab-7, Fab-7 and Fab-8). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can 'jump over' intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation.

Boundary Bypass Activity in the Abdominal-B Region of the Drosophila Bithorax Complex is Position Dependent and Regulated.

Expression of Abdominal-B ( Abd-B ) in abdominal segments A5 - A8 is controlled by four regulatory domains, iab-5 - iab-8 . 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 ( Mcp , Fab-7 , Fab-7 and Fab-8 ). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can "jump over" intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation. Summary Statement: Boundaries separating Abd-B regulatory domains block crosstalk between domains and mediate their interactions with Abd-B . The latter function is location but not orientation dependent.

lncRNA read-through regulates the BX-C insulator Fub-1.

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

Application of the 3C Method to Study the Developmental Genes in Drosophila Larvae.

A transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in Drosophila larvae. We critically analyzed the existing protocols and offered our own solutions and the optimized protocol to overcome limitations. To demonstrate the efficiency of our procedure, we studied the spatial organization of the developmental locus Dad in 3rd instar Drosophila larvae. Differences in locus conformation were found between embryonic cells and living wild-type larvae. We also observed the establishment of novel regulatory interactions in the presence of an adjacent transgene upon activation of its expression in larvae. Our work fills the gap in the application of the 3C method to Drosophila larvae and provides a useful guide for establishing 3C on an animal model.

Investigation of the Basic Steps in the Chromosome Conformation Capture Procedure.

A constellation of chromosome conformation capture methods (С-methods) are an important tool for biochemical analysis of the spatial interactions between DNA regions that are separated in the primary sequence. All these methods are based on the long sequence of basic steps of treating cells, nuclei, chromatin, and finally DNA, thus representing a significant technical challenge. Here, we present an in-depth study of the basic steps in the chromatin conformation capture procedure (3С), which was performed using Drosophila Schneider 2 cells as a model. We investigated the steps of cell lysis, nuclei washing, nucleoplasm extraction, chromatin treatment with SDS/Triton X-100, restriction enzyme digestion, chromatin ligation, reversion of cross-links, DNA extraction, treatment of a 3C library with RNases, and purification of the 3C library. Several options were studied, and optimal conditions were found. Our work contributes to the understanding of the 3C basic steps and provides a useful guide to the 3C procedure.

Molecular Basis of the Function of Transcriptional Enhancers.

Transcriptional enhancers are major genomic elements that control gene activity in eukaryotes. Recent studies provided deeper insight into the temporal and spatial organization of transcription in the nucleus, the role of non-coding RNAs in the process, and the epigenetic control of gene expression. Thus, multiple molecular details of enhancer functioning were revealed. Here, we describe the recent data and models of molecular organization of enhancer-driven transcription.

Evolution of Regulated Transcription.

The genomes of all organisms abound with various cis-regulatory elements, which control gene activity. Transcriptional enhancers are a key group of such elements in eukaryotes and are DNA regions that form physical contacts with gene promoters and precisely orchestrate gene expression programs. Here, we follow gradual evolution of this regulatory system and discuss its features in different organisms. In eubacteria, an enhancer-like element is often a single regulatory element, is usually proximal to the core promoter, and is occupied by one or a few activators. Activation of gene expression in archaea is accompanied by the recruitment of an activator to several enhancer-like sites in the upstream promoter region. In eukaryotes, activation of expression is accompanied by the recruitment of activators to multiple enhancers, which may be distant from the core promoter, and the activators act through coactivators. The role of the general DNA architecture in transcription control increases in evolution. As a whole, it can be seen that enhancers of multicellular eukaryotes evolved from the corresponding prototypic enhancer-like regulatory elements with the gradually increasing genome size of organisms.

The insulator functions of the Drosophila 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 Drosophila 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). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.