Larisa Melnikova

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.

‘Insulator bodies’ are aggregates of proteins but not of insulators

Chromatin insulators are thought to restrict the action of enhancers and silencers. The best‐known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator‐related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are ‘insulator bodies’ of many Su(Hw)–insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q‐rich domain supports the function of Su(Hw)‐dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)‐marked nuclear speckles; conversely, the latter accumulate another (C‐truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so‐called ‘insulator bodies’.

Study of Long-Distance Functional Interactions between Su(Hw) Insulators That Can Regulate Enhancer-Promoter Communication in Drosophila melanogaster

ABSTRACT The Su(Hw) insulator found in the gypsy retrotransposon is the most potent enhancer blocker in Drosophila melanogaster. However, two such insulators in tandem do not prevent enhancer-promoter communication, apparently because of their pairing interaction that results in mutual neutralization. Furthering our studies of the role of insulators in the control of gene expression, here we present a functional analysis of a large set of transgenic constructs with various arrangements of regulatory elements, including two or three insulators. We demonstrate that their interplay can have quite different outcomes depending on the order of and distance between elements. Thus, insulators can interact with each other over considerable distances, across interposed enhancers or promoters and coding sequences, whereby enhancer blocking may be attenuated, cancelled, or restored. Some inferences concerning the possible modes of insulator action are made from collating the new data and the relevant literature, with tentative schemes illustrating the regulatory situations in particular model constructs.

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3′UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari–Wari, Wari–gypsy or 1A2–Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.

Drosophila telomeres: the non-telomerase alternative

In most eukaryotes, telomeres are composed of simple repetitive sequences renewable by telomerase. By contrast, Drosophila telomeres comprise arrays of non-LTR retrotransposons HeT-A, TART, and TAHRE belonging to three different families. However, closer inspection reveals that the two quite different telomere systems share quite a few components and regulatory circuits. Here we present the current knowledge on Drosophila telomeres and discuss the possible mechanisms of telomere length control.

Zeste can facilitate long-range enhancer-promoter communication and insulator bypass in Drosophila melanogaster.

The looping model of enhancer–promoter interactions predicts that these specific long-range interactions are supported by a certain class of proteins. In particular, the Drosophila transcription factor Zeste was hypothesized to facilitate long-distance associations between enhancers and promoters. We have re-examined the role of Zeste in supporting long-range interactions between an enhancer and a promoter using the white gene as a model system. The results show that Zeste binds to the upstream white promoter region and the enhancer that is responsible for white activation in the eyes. We have confirmed the previous finding that Zeste is not required for the activity of the eye enhancer and the promoter when they are located in close proximity to each other. However, inactivation of Zeste markedly affects the enhancer–promoter communication in transgenes when the eye enhancer and the white promoter are separated by a 3-kb spacer or the yellow gene. Zeste is also required for insulator bypass by the eye enhancer. Taken together, these results show that Zeste can support specific long-range interactions between enhancers and promoters.

Drosophila gypsy insulator and yellow enhancers regulate activity of yellow promoter through the same regulatory element

There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of the enhancer–promoter interaction in yellow. In this paper, we have found that the yellow sequence from −100 to −69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4)u1 background. These finding suggest that proteins bound to the −100 to −69 sequence are essential for communication between the yellow promoter and upstream regulatory elements.

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

EAST affects the activity of Su(Hw) insulators by two different mechanisms in Drosophila melanogaster

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.

MOD(MDG4)-64.2 protein, isoform of MOD(MDG4) loci, directly interacts with the Tweedle protein family of Drosophila melanogaster.

225 Transcription regulation in higher eukaryotes is ensured by the interaction between intricate protein complexes that are formed on the sequences of genomic regulatory elements. The promoters that define the transcription initiation and its basic level as well as the cisregulatory elements, which either enhance (enhancers) or attenuate (silencers) the basic level of transcription, have been studied sufficiently well. Unlike them, insulators are a relatively new class of regulatory elements. Initially, insulators described as regulatory elements that (1) are located between the enhancer and promoter and can block the interaction between them, without affecting their functionality and (2) exhibit barrier properties, i.e., prevent the inactivating effect of heterochromatin on the trann scription of the gene surrounded by them. It was then found that the role of insulators in the regulation of transcription is not unambiguous [1]. Currently, the mechanism of action of insulators and their role in the genome are not understood completely. There are sevv eral models of functioning of insulators, each of which has its advantages and disadvantages. Unfortunately, none of them is universal because it is not supported by sufficient experimental data. The best studied insulator is the Su(Hw) insulator of D. melanogaster [2, 3]. One of the main compoo nents of the Su(Hw))dependent insulator complex is the Mod(mdg4) protein. It is required for the insulator function and is connected to it through the interaction with the Su(Hw) protein [4, 5]. The mod(mdg4) gene was identified in many various genetic experiments. It was independently identified in the screening of mutations that affect PEV, alter the properties of insuu lator sequences, and influence the development of nervous cells, conjugation of chromosomes during meiosis, and apoptosis [6]. The molecular analysis of the mod(mdg4) locus showed that it encodes a family consisting of at least of 26 protein isoforms. It was shown that, to produce mature transcripts, this locus uses the mechanism of transssplicing of mRNA. The NNterminus of all splice variants contains the BTB/POZ domain and a glutaminee rich region [7, 8]. The BTB/POZ domain provides the interaction between proteins and the formation of homodimers, heterodimers, and oligomers [9, 10]. However, all splice variants differ in their CCterminal domains [4, 5]. The majority of CCtermini contain the conserved protein motif Cys 2 His 2 , which is called FLYWCH [4–6]. Mutations that affecting the mod(mdg4) gene region that encodes the sequence common for all isoforms of the Mod(mdg4) protein …