Pavel Georgiev

Heterochromatin Protein 1 Is Involved in Control of Telomere Elongation in Drosophila melanogaster

ABSTRACT Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.

Insulators, Not Polycomb Response Elements, Are Required for Long-Range Interactions between Polycomb Targets in Drosophila melanogaster

ABSTRACT The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb “bodies” or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.

The Mcp Element from the bithorax Complex Contains an Insulator That Is Capable of Pairwise Interactions and Can Facilitate Enhancer-Promoter Communication

ABSTRACT Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3′ cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

Attachment of HeT-A Sequences to Chromosomal Termini in Drosophila melanogaster May Occur by Different Mechanisms

ABSTRACT Drosophila telomeres contain arrays of the retrotransposonlike elements HeT-A and TART. Their transposition to broken chromosomal termini has been implicated in chromosome healing and telomere elongation. The HeT-Aelement is attached by its 3′ end, which contains the promoter. To monitor the behavior of HeT-A elements, we used the yellow gene with terminal deficiencies consisting of breaks in theyellow promoter region that result in they-null phenotype. Attachment of the HeT-Aelement provides the promoterless yellow gene with a promoter that activates yellow expression in bristles. The frequency of HeT-A transpositions to the yellowterminal deficiency depends on the genotype of the line and varies from 2 × 10−3 to less than 2 × 10−5. Loss of the attached HeT-A due to incomplete replication at the telomere leads to inactivation of yellow expression, which is restored by attachment of a new HeT-A element upstream of yellow. New HeT-A additions occur at a frequency of about 1.2 × 10−3. Short DNA attachments are generated by gene conversion using the homologous telomeric sequences as templates. Longer DNA attachments are generated either by conventional transposition of an HeT-A element to the chromosomal terminus or by recombination between the 3′ terminus of telomeric HeT-A elements and the receding end ofHeT-A attached to the yellow gene.

An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila

The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.

Study of the Functional Interaction between Mcp Insulators from the Drosophila bithorax Complex: Effects of Insulator Pairing on Enhancer-Promoter Communication

ABSTRACT Boundary elements have been found in the Abd-B 3′ cis-regulatory region, which is subdivided into a series of iab domains. Previously, a 340-bp insulator-like element, M340, was identified in one such 755-bp Mcp fragment linked to the PcG-dependent silencer. In this study, we identified a 210-bp core that was sufficient for pairing of sequence-remote Mcp elements. In two-gene transgenic constructs with two Mcp insulators (or their cores) surrounding yellow, the upstream yeast GAL4 sites were able to activate the distal white only if the insulators were in the opposite orientations (head-to-head or tail-to-tail), which is consistent with the looping/bypass model. The same was true for the efficiency of the cognate eye enhancer, while yellow thus isolated in the loop from its enhancers was blocked more strongly. These results indicate that the relative placement and orientation of insulator-like elements can determine proper enhancer-promoter communication.

Making connections: Insulators organize eukaryotic chromosomes into independent cis-regulatory networks

Insulators play a central role in subdividing the chromosome into a series of discrete topologically independent domains and in ensuring that enhancers and silencers contact their appropriate target genes. In this review we first discuss the general characteristics of insulator elements and their associated protein factors. A growing collection of insulator proteins have been identified including a family of proteins whose expression is developmentally regulated. We next consider several unexpected discoveries that require us to completely rethink how insulators function (and how they can best be assayed). These discoveries also require a reevaluation of how insulators might restrict or orchestrate (by preventing or promoting) interactions between regulatory elements and their target genes. We conclude by connecting these new insights into the mechanisms of insulator action to dynamic changes in the three‐dimensional topology of the chromatin fiber and the generation of specific patterns of gene activity during development and differentiation.

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs–scs, scs’–scs’, 1A2–1A2 and Wari–Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5–Su(Hw), dCTCF–Su(Hw), or dCTCF–Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

Chromatin insulators and long-distance interactions in Drosophila

Data on long‐distance enhancer‐mediated activation of gene promoters and complex regulation of gene expression by multiple enhancers have prompted the hypothesis that the action of enhancers is restricted by insulators. Studies with transgenic lines have shown that insulators are responsible for establishing proper local interactions between regulatory elements, but not for defining independent transcriptional domains that restrict the activity of enhancers. It has also become apparent that enhancer blocking is only one of several functional activities of known insulator proteins, which also contribute to the organization of chromosome architecture and the integrity of regulatory elements.

TAFII40 protein is encoded by the e(y)1 gene: biological consequences of mutations.

ABSTRACT The enhancer of yellow 1 gene, e(y)1, ofDrosophila melanogaster has been cloned and demonstrated to encode the TAFII40 protein. The e(y)1 gene is expressed in females much more strongly than in males due to the accumulation of e(y)1 mRNA in the ovaries. Two differente(y)1 mutations have been obtained. Thee(y)1ul mutation, induced by the insertion ofStalker into the coding region, leads to the replacement of 25 carboxy-terminal amino acids by 17 amino acids encoded by theStalker sequences and to a decrease of thee(y)1 transcription level. The latter is the main cause of dramatic underdevelopment of the ovaries and sterility of females bearing the e(y)1 mutation. This follows from the restoration of female fertility upon transformation ofe(y)1u1 flies with a construction synthesizing the mutant protein. The e(y)1P1 mutation induced by P element insertion into the transcribed nontranslated region of the gene has almost no influence on the phenotype of flies. However, in combination with thephP1 mutation, which leads to a strongP element-mediated suppression of e(y)1transcription, this mutation is lethal. Genetic studies of thee(y)1u1 mutation revealed a sensitivity of theyellow and white expression to the TAFII40/e(y)1 level. The su(Hw)-binding region,Drosophila insulator, stabilizes the expression of thewhite gene and makes it independent of thee(y)1u1 mutation.