ISSN 1607�6729, Doklady Biochemistry and Biophysics, 2009, Vol. 429, pp. 293–295. © Pleiades Publishing, Ltd., 2009.Original Russian Text © A.P. Kotnova, V.B. Salenko, N.V. Lyubomirskaya, Y.V. Ilyin, 2009, published in Doklady Akademii Nauk, 2009, Vol. 429, No. 1, pp. 120–122.

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Transposable genetic elements constitute a consid�erable part of eukaryotic genome and have a markedeffect on its function and structurization [1]. The pres�ence of a great number of highly diverged copies of dif�ferent transposable elements in the heterochromatinof eukaryotes has long remained a mystery and wasmost often considered as a peculiar “cemetery” of ret�rotransposons. However, recent studies showed thatthese heterochromatin regions harbor the clusters oftransposable elements producing short RNAs, whichmay be then involved in the elimination of transcriptsof active euchromatic copies of transposable elements[2]. There is another relevant problem that requiresspecial attention of researchers—the involvement ofassociations of transposable elements in the formationof chromatin architecture. It was shown that sometransposable elements (in particular, the MDG4endogenous retrovirus of Drosophila melanogaster,which is also known as gypsy) contain regulatorysequences, such as insulators, which can influence thefunctioning of genes located at large distances fromthe transposable element [3, 4]. However, the questionon the effect of transposable element clusters occur�ring in the genome on the chromatin conformationand their role in the formation of epigenetic structuresthat can affect gene function without changing the pri�mary structure of genomic DNA remains to beanswered.

In this study, we revealed two regions in the D. mel�anogaster genome containing inverted repeats repre�sented by fragments of transposable elements. Possibleinvolvement of such structures in the regulation andfunctioning of the genome is discussed.

In the past decade, the D. melanogaster strain G32has been actively studied in our laboratory. Thegenome of this strain, along with the active and inac�

tive MDG4 (gypsy) variants, contains a hybrid variant[5, 6]. The genomic library of D. melanogaster strainG32 contains 24 different MDG4 copies, the majorityof which are located in the heterochromatin [6]. Adetailed structural analysis of these copies and theirgenomic environment provides a unique opportunityto study heterochromatin structure within one strainand compare it with the sequences available fromdatabases.

The sequencing of the clones from the genomiclibrary of D. melanogaster strain G32 that containdiverged MDG4 (gypsy) copies showed that one clone,named 40A, contained a sequence with invertedrepeats represented by fragments of different transpos�able elements. To determine the location of the clonedsequences, we performed the search for homologoussites in the FlyBase database of the Drosophilagenome. As a result, clone 40A was found to be local�ized to the X�chromosome heterochromatic region(X Het: 151118–135955). In addition, successfullocalization of clone 4.2 of the D. melanogaster strainG32 to the heterochromatin of the left arm of chromo�some 3 (3LHet: 1614362–1623973) made it possibleto find one more fragment in the FlyBase database ofthe Drosophila genome, containing three repeats oftransposable element clusters—two direct and oneinverted.

Figure 1 shows the schematic structure of clone40A (15164 bp), which is represented primarily byfragments of different transposable elements. Frag�ments of the same element adjoining one another areunited. Of special interest is the presence of twoinverted repeats, which are indicated with thin arrowsbelow the scheme (forward repeat, 5151 bp; reverserepeat, 5168 bp). Each repeat is represented by oppo�sitely directed highly rearranged transposable ele�ments BS (fragments 1749–2010, 2098–3564, 3785–4296, and 4467–5103 or 4467–5116 are united withinone white arrow) and gypsy (fragments 934–1004,1202–1660, and 4286–4398 are united within onegray arrow; fragments 4436–4505 and 4615–4912,

Structural Organization of Heterochromatinin Drosophila melanogaster: Inverted Repeats

of Transposable Element ClustersA. P. Kotnova, V. B. Salenko, N. V. Lyubomirskaya, and Academician Y. V. Ilyin

Received June 2, 2009

DOI: 10.1134/S1607672909060027

Engelhardt Institute of Molecular Biology,Russian Academy of Sciences,ul. Vavilova 32, Moscow, 119991 Russia

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within the other gray arrow), as well as includes a smallregion of the G element (fragment 4041–4181),located between the repeats and carrying multipledeletions (shown with thick black arrows). Thenumeration of nucleotides is given according to thecanonical sequence for each of the mentioned trans�posable elements. The identity of repeats was veryhigh: over a 5�kb region they only differed in singlesubstitutions and several deletions/insertions of singlenucleotides.

Figure 2 shows a detailed structure of clone 4.2,containing the fragments of elements X, MDG4(gypsy), Doc, and gypsy12. Its length was 9612 bp.When determining the location of this fragment in theDrosophila genome, it was found that the sequencefrom the database that adjoins clone 4.2 contains two

more repeats of the fragment of this clone (forwardand inverted), with slight variations. The schematicrepresentation of this region, located in the hetero�chromatic regions of chromosome 3 (3LHet:1614362–1646000) is shown in Fig. 3. Fragments ofthe same element adjoining one another are united.

As can be seen from Fig. 2, the repeated region(5676 bp long) present in clone 4.2 is represented bythe following fragments of transposable elements: Xelement (880–953, 1711–2521, and 2550–3616) andgypsy (823–896, 932–1018, 1202–1923, 2772–2886,3536–3789, 3824–4114, 4194–4912, 4952–5400,and 5676–5808). The numeration of nucleotides isgiven according to the canonical sequence for each ofthe mentioned transposable elements. A forwardrepeat 5943 bp long is located approximately 13 kb

BS (1749–5103)

gypsy (934–4398)

gypsy (4436–4912)

G (3867–4181)

gypsy (4934–5212) G (653–3651)

G (4041–4181)

gypsy (4436–4912)

gypsy (934–4398)

BS (1749–5116)

1 kb

Repeat 1 Repeat 2

X (4132–4290)

X (880–953)

X (1711–2521)

X (2550–3616)

gypsy (932–1018)gypsy (1202–1923)

gypsy (2772–2886)

gypsy (3536–3789)

gypsy (3824–4114) gypsy (4194–4912)

gypsy (4952–5400)gypsy (5676–5808)

Doc (2503–2840)

gypsy12 (2750–2824)gypsy12 (3276–3800)

gypsy12 (3991–4453)gypsy12 (4831–5092)

gypsy12 (5128–5492)

1 kbRepeat 1

Fig. 1. Clone 40A structure. Schematic representation of clone 40A from D. melanogaster strain G32. Fragments of the samediverged transposable element adjoining each other are united. Thick white arrows indicate the BS element fragments; grayarrows, the MDG4 (gypsy) fragments; and black arrows, the G element fragments. Thin arrows below the scheme indicateinverted repeats. Here and in Figs. 2 and 3, numerals in parentheses correspond to the serial numbers of nucleotides in thesequence of each transposable element.

Fig. 2. Clone 4.2 structure. Detailed structure of clone 4.2 from the D. melanogaster strain G32. Thick white arrows indicate theX element fragments; gray arrows, the MDG4 (gypsy) fragments; and black arrows, the gypsy12 fragments. The fragment of theDoc element is crosshatched with oblique lines. The thin arrow below the scheme indicates one of three repeats in the hetero�chromatin in chromosome, which is present in the clone.

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from it (Fig. 3). It is slightly different. First, the frag�ment of the X element (880–953) is absent in it. Sec�ond, there is one more small MDG4 (gypsy) repeat,which, along with the sequence 1202–1923, containssequence 1202–1850 immediately upstream of theformer (i.e., this region underwent dimerization);third, it ends with a more extended MDG4 (gypsy)fragment (5676–6558 instead of 5676–5808 inclone 4.2). There are some more insignificant dele�tions and substitutions. This repeat is immediately fol�lowed by the third, inverted repeat 5490 bp long, themain distinction of which from the cluster from clone4.2 is the absence of the MDG4 (gypsy) fragment5676–5808 and truncated variant of the previousregion (the inverted repeat contains sequence 4958–5317 instead of sequence 4952–5400 in clone 4.2).

The structures discovered by us in the Drosophilagenome, containing inverted repeats within transpos�able element clusters are undoubtedly of interest. Thepresence of such sequences in the genome of theD. melanogaster strain G32 as well as in the genomefrom the FlyBase database is indicative of their con�served nature, which is an indirect evidence for theirimportant functions. It is known that both sense andantisense short RNAs are substrates of different groupsof enzymes involved in cascades of RNA silencingreactions [2]. The presence in the genome ofsequences able to simultaneously produce both shortRNA variants led us to assume that these regions mayfunction as a much more potent blocker of transposi�tion of mobile elements than the extended hetero�chromatic clusters devoid of such repeats. In addition,such structures may be involved in the formation ofstable epigenetic relationships. The MDG4 (gypsy)

endogenous retrovirus contains an insulator, whosefragment is present in inverted repeats of the discov�ered transposable element clusters. The role of MDG4(gypsy) insulator in the formation of epigenetic struc�tures is already confirmed; however, the mechanismsof formation of insulator bodies have been insuffi�ciently studied and are debated in the world scientificcommunity. The origin of the inverted clusters oftransposable elements remains to be established.

ACKNOWLEDGMENTS

This study was supported by the program of thePresident of the Russian Federation for support ofyoung candidates of science (project no. MK�5439.2008.4), the program “Leading ScientificSchools” (project no. NSh�2994.2008.4), and theRussian Foundation for Basic Research (projectnos. 08�04�00227�a and 09�04�12172�ofi_M).

REFERENCES

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2. Brennecke, J., Aravin, A.A., Stark, A., et al., Cell, 2007,vol. 128, pp. 1089–1103.

3. Gdula, D.A., Gerasimova, T.I., and Corces, V.G., Proc.Natl. Acad. Sci. USA, 1996, vol. 93, pp. 9378–9383.

4. Dorsett, D., Curr. Opin. Genet. Dev., 1999, vol. 9,pp. 505–514.

5. Lyubomirskaya, N.V., Smirnova, J.B., Razorenova,O.V., et al., Mol. Gen. Genom., 2001, vol. 265, pp. 367–374.

6. Salenko, V.B., Kotnova, A.P., Karpova, N.N., et al.,Mol Gen. Genom., 2008, vol. 279, no. 5, pp. 463–472.

X (4132–4290)X (880–953)

X (1711–3616)gypsy (923–5808)

Doc (2503–2840)

gypsy12 (3276–7886)Doc (2859–3595)

Doc (2963–3038)

gypsy12 (5584–6613)1360 (51–569) 1360 (3114–3409)

gypsy12 (2122–3668)X (4427–4654)

X (1711–3616)gypsy (823–6558)

gypsy (823–5317)X (1711–3616)

X (4132–4713)X (880–953)

5 kb

Repeat 1 Repeat 2 Repeat 3

Clone 4.2

Fig. 3. Structure of the Drosophila genome region 3LHet: 1614362–1646000 taken from the Flybase database. Schematic repre�sentation of the Drosophila genome region 3LHet: 1614362–1646000 taken from the Flybase database. Fragments of the samediverged transposable element adjoining each other are united. Thick white arrows indicate the X element fragments; gray arrows,the MDG4 (gypsy) fragments; and black arrows, the gypsy12 fragments. The fragments of the Doc element and element 1360 arecrosshatched with oblique and horizontal lines, respectively. Thin arrows below the scheme indicate repeats. The line below thescheme indicates the position of clone 4.2 from the D. melanogaster strain G32.