help button home button Genetics J Gen Phys
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Originally published as Genetics Published Articles Ahead of Print on December 6, 2006.

Genetics, Vol. 175, 609-620, February 2007, Copyright © 2007
doi:10.1534/genetics.106.062133

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
genetics.106.062133v1
175/2/609    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Demakova, O. V.
Right arrow Articles by Zhimulev, I. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Demakova, O. V.
Right arrow Articles by Zhimulev, I. F.

The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

Olga V. Demakova, Galina V. Pokholkova, Tatyana D. Kolesnikova, Sergey A. Demakov, Eugenia N. Andreyeva, Elena S. Belyaeva and Igor F. Zhimulev1

Laboratory of Molecular Cytogenetics, Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk 630090, Russia

1 Corresponding author: Institute of Cytology and Genetics, Russian Academy of Sciences, 10 Lavrentiev Ave., Novosibirsk 630090, Russia.
E-mail: zhimulev{at}bionet.nsc.ru

Manuscript received June 18, 2006. Accepted for publication November 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26–h29) is the only one within the chromocenter to form a big "puff"-like structure. The "puffed" heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.

IN Drosophila melanogaster, chromatin is organized generally into two distinct types: euchromatin (eu) and heterochromatin. Condensed throughout most of the cell cycle, the heterochromatin is located predominantly around centromeres. Enrichment of repetitive DNA sequences, low density of unique genes, and late replication in S-phase are the primary features of the heterochromatin that are established early in development (DIMITRI et al. 2005). Additionally, in polytene chromosomes, pericentric heterochromatin (PH) regions are highly underreplicated and are fused to form a single chromocenter (reviewed by GATTI and PIMPINELLI 1992; ZHIMULEV 1998; GREWAL and ELGIN 2002). The presence of histone variants and specifically modified histones as well as the recruitment of a number of PH-associated nonhistone proteins is considered to play a central role in the establishment and maintenance of the repressive higher-order chromatin structure of PH (TURNER et al. 1992; RICHARDS and ELGIN 2002; KAMAKAKA and BIGGINS 2005).

Current models for heterochromatin formation suggest that it is a multi-step process (SWAMINATHAN et al. 2005). Methylation of histone H3K9 by histone methyltransferase (HMTase) SU(VAR)3-9 is a key step in creating binding sites for HP1, which associates with PH through its chromodomain (REA et al. 2000; BANNISTER et al. 2001; LACHNER et al. 2001; NAKAYAMA et al. 2001). The predominant targeting of SU(VAR)3-9 to the polytene chromocenter, in turn, is dependent on functional HP1 protein (SCHOTTA et al. 2002). The latter is known to interact with a large number of other proteins and, thus, to contribute to various cell processes (EISSENBERG and ELGIN 2000; LI et al. 2002). Recently, HP1 was shown to target and cooperate with another heterochromatin-specific protein, SU(VAR)3-7 (CLEARD et al. 1997; DELATTRE et al. 2000; SPIERER et al. 2005), which has affinity for DNA (CLEARD and SPIERER 2001; PERRINI et al. 2004). Additionally, interaction of SU(VAR)3-7 with SU(VAR)3-9 has been revealed by yeast two-hybrid assay (SCHOTTA et al. 2002) and in vivo (DELATTRE et al. 2004). Thus, the targeting and/or proper distribution of these nonhistone proteins in chromatin appear largely interdependent.

Eu–heterochromatin dichotomy in classification of Drosophila chromatin is, however, only a simplification. First, numerous intercalary heterochromatin (IH) regions are distributed along the euchromatic arms. The IH displays features characteristic of PH such as late replication in S-phase, underreplication in polytene chromosomes, and transcriptional inactivity in salivary glands (ZHIMULEV and BELYAEVA 2003). IH regions are now considered to be sites of epigenetic silencing of specific clusters of unique genes (BELYAKIN et al. 2005).

Second, special eu–heterochromatin transition zones can be distinguished, which probably evolved in wild-type chromosomes to separate two types of chromatin state. These zones appear to be of dual nature, displaying both the eu and the heterochromatic properties (DEVLIN et al. 1990; MITCHELSON et al. 1993; BERGHELLA and DIMITRI 1996; MOSHKIN et al. 2002; GREIL et al. 2003). Furthermore, it is widely accepted that genes require their native chromosomal environment to be expressed properly. Eu–heterochromatin junctions, newly arisen as a result of chromosomal rearrangements, often lead to drastic local changes in chromatin organization, which can result in stochastic transcriptional silencing of mislocated genes (reviewed by WEILER and WAKIMOTO 1995; ELGIN 1996). This phenomenon, termed position-effect variegation (PEV), correlates with spreading of heterochromatin proteins along an adjacent euchromatic region, which acquires a heterochromatin-like structure and becomes late replicating (reviewed by BELYAEVA et al. 1993; ZHIMULEV 1998; EBERT et al. 2004).

Finally, the PH is shown to be heterogeneous itself at different levels of its organization. A series of distinct blocks revealed within mitotic PH by differential staining (GATTI et al. 1994) as well as the "islands" of complex DNA sequences distributed along extensive areas of simple satellites (LE et al. 1995, SUN et al. 2003) reflect the complexity of PH structure. Heterochromatic regions also differ in their ability to induce PEV (reviewed in ZHIMULEV 1998). Some nonhistone proteins were demonstrated to associate differentially with PH components: the AT-hook protein D1 plays a direct role in assembly of AT-rich heterochromatin, specifically binding to some satellite repeats (AULNER et al. 2002). In D. melanogaster, CID protein replaces the histone H3 in centromeres and is essential for their function (AHMAD and HENIKOFF 2002). Additionally, the SUUR protein, mutational loss of which is known to suppress underreplication of heterochromatic regions, affects all IH regions, although PH is affected only partially (BELYAEVA et al. 1998; BELYAKIN et al. 2005). However, our understanding of how characteristic heterochromatin proteins contribute to heterogeneous structure of PH is far from complete.

SU(VAR)3-9, HP1, SU(VAR)3-7, and SUUR are encoded by genes that have been identified as dominant modifiers of PEV (TSCHIERSCH et al. 1994; CLEARD et al. 1997; BELYAEVA et al. 2003). Su(var)3-9 dominates the PEV modifier effects of Su(var)2-5 (HP1) and Su(var)3-7 genes (SCHOTTA et al. 2002). The silencing potential of SU(VAR)3-9 clearly depends on its HMTase activity, which controls most H3K9 di- and trimethylation at PH (EBERT et al. 2004). To gain insight into the role of SU(VAR)3-9 in vivo, we investigated the consequences of loss or reduction of its HMTase activity for the structure and replication of heterochromatin in salivary gland polytene chromosomes, focusing on the distal heterochromatin of the X chromosome (distal Xh). Strong local decompaction of the distal Xh (pseudopuff formation) was found in the absence of either SU(VAR)3-9 or HP1, which is accompained by changes in the replication state of heterochromatin and a set of heterochromatin-specific proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Fly stocks and constructs:
Flies were reared on standard medium at 25°. Su(var)3-9 alleles used were Su(var)3-907, Su(var)3-906, and Su(var)3-922 (EBERT et al. 2004). The heteroallelic combinations Su(var)3-907/Su(var)3-906 and Su(var)3-922/Su(var)3-906 were identified in a progeny from the cross Su(var)3-9mut/TM6C x Su(var)3-906 by the absence of the Tubby marker. The Suppressor of Underreplication (SuUR) mutation was originally detected in the In(1)scV2 stock (BELYAEVA et al. 1998). Double-mutant flies SuUR Su(var)3-906 were obtained by recombining these mutations from ru h SuUR and e ro Su(var)3-906 chromosomes. 4xSuUR+ stock contains two endogenuous and two transgenic copies of the SuUR+ gene (BELYAEVA et al. 2003). A series of stocks were constructed in which In(1)wm4h was combined with 3L chromosomes containing the SuUR or Su(var)3-906 mutation or both. In(1)scV2, In(1)sc4, and In(1)sc8 were introduced only on the SuUR Su(var)3-906 background. We generated the Su(var)3-906 Sgs3-GAL4 stock by recombining the Sgs3-GAL4 transgene (CHERBAS et al. 2003) onto the Su(var)3-906 background. Effect of Su(var)3-9 overexpression was analyzed in GAL4>UAS system (BRAND and PERRIMON 1993) in a progeny from the cross UAS-Su(var)3-9+-EGFP; Su(var)3-906 x Su(var)3-906 Sgs3-GAL4. Description of balancers, mutations, and chromosomes not specifically mentioned here is given in FlyBase (DRYSDALE et al. 2005).

Cytology:
Fluorescence in situ hybridization (FISH) on polytene chromosomes was performed as described in KORYAKOV et al. (2003). DNA clones were labeled with biotin-16-dUTP (Roche) in random-primed polymerase reaction with a Klenow fragment; a 6.4-kb BamHI–XbaI fragment of the su(f) gene (YAMAMOTO et al. 1990); a 0.9-kb HindIII fragment of the 28S rRNA gene (TAUTZ et al. 1988); a 1.15-kb BglII fragment from Stellate repeats (TULIN et al. 1997); and c23, a 45-kb DNA fragment from the white locus (PIRROTTA et al. 1983) were used.

Immunostaining was performed as described in CZERMIN et al. (2002). The primary antibody dilutions used were as follows: rabbit polyclonal anti-SUUR (E-45), 1:50; mouse monoclonal anti-HP1 (CA19), 1:80; rabbit polyclonal HP2, 1:600; rabbit polyclonal SU(VAR)3-7, 1:400; rabbit polyclonal anti-H4AcK12 (Serotec, Oxford), 1:100; mouse monoclonal Z4, 1:30; rabbit polyclonal JIL-1, 1:100; rabbit polyclonal H3Me3K4 (Abcam), 1:100; monoclonal H14 mouse IgM antibodies against the Ser5-phosphorylated CTD of RNA polymerase II–PolIIo (Covance), 1:50; rabbit polyclonal anti-MSL2, 1:50. The squashes were incubated with secondary FITC or rhodamine-labeled goat anti-rabbit and anti-mouse IgG-specific conjugates (Abcam, 1:200) or with anti-mouse IgM–FITC conjugates [Sigma (St. Louis), 1:250]. The labeling signal by SU(VAR)3-7 antibodies was enhanced by using rabbit FITC anti-goat IgG-specific conjugates (Sigma, 1:200).

Labeling of polytene chromosomes with 5'-iodo-2'-deoxyuridine (IdU, Sigma) was performed as described in ANDREYEVA et al. (2005). To ensure reproducibility of labeling, immunostaining and IdU incorporation were performed three to five times with 5–10 slides in each experiment. Preparations of salivary gland chromosomes stained with acetic orcein were made by standard method.

Pulsed-field electrophoresis and Southern blot hybridization:
Salivary gland nuclei isolated from the third instar larvae were embedded into agarose and DNA was treated in agarose inserts as described in KARPEN and SPRADLING (1990). The DNA samples were digested with NotI and fractionated by contour-clamped homogeneous electric field pulsed-field gel electrophoresis on a Bio-Rad (Hercules, CA) DRII apparatus. Electrophoresis conditions were 6 V/cm at 14° and 3–30 sec linearly ramped pulses for 16 hr in 0.5x TBE.

After pulsed-field gel electrophoresis, DNA was transferred to the Hybond-N filter (Amersham-Pharmacia-Biotech) and analyzed by Southern blot hybridization as described earlier (KARPEN and SPRADLING 1990).

Cosmid c23 DNA was labeled with [32P]dATP to specific activities of ~109 cpm/µg by random priming and was used as a probe for hybridization. Hybridization signals were analyzed qualitatively using Kodak XAR5 film.

Positions of NotI restriction sites within the white locus were determined according to the Berkeley Drosophila Genome Project, r 4.2 (http://www.fruitfly.org).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Loss or reduction of SU(VAR)3-9 HMTase activity affects morphology and compaction state of pericentric heterochromatin:
To investigate the role of SU(VAR)3-9 in establishment of large silent chromatin domains in vivo, we tested whether loss or decrease in its HMTase activity would affect PH morphology in salivary gland polytene chromosomes. To this end, we made use of the null-allele Su(var)3-906 and of two mutant alleles providing reduced HMTase activity levels, Su(var)3-907 and Su(var)3-922 (EBERT et al. 2004). We found that the chromocenter is unusually large and appears more decondensed in the Su(var)3-9 mutants (Figure 1, A and B).


Figure 1
View larger version (72K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 1.— Effect of Su(var)3-9 and Su(var)2-5 mutations on the morphology of PH regions of the X chromosome. (A) Chromosomal pericentric regions are fused into a compact chromocenter in wild-type strain. Dashed line contours the chromocenter. (B) In Su(var)3-906 homozygotes, the chromocenter looks larger and more decondensed. Dashed line delimits the chromocenter. (C) In wm4/Y, section 20 of the X chromosome is represented by the single band 20A1-2 and a compact block of chromatin, which is composed of a part of heterochromatin-like region 20 joined to the euchromatic region 3C. (D) In wm4/Y; Su(var)3-906 homozygotes, section 20 becomes finely banded. A large pseudopuff (PP) is present in the area of the 20F-3C junction. (E) A pseudopuff in region 20 of wm4/Y; Su(var)3-906 mutants is dramatically reduced in larvae with the Su(var)3-9 transgene ectopically overexpressed using the GAL4/UAS system. (F) In the absence of zygotic HP1 in wm4; Su(var)2-505/Su(var)2-503 larvae, the PP of moderate sizes develops. Bar, 5 µm.

 
To gain further insight into the way in which SU(VAR)3-9 contributes to the characteristic condensation of distinct PH regions, we precisely analyzed section 20 of the X chromosome, which contains both the eu–heterochromatin transition zone and the distal part of the Xh. This region is enriched with repetitive DNA and replicates late in S-phase (for review see ZHIMULEV 1998). Similarly to euchromatin, region 20 harbors many unique genes that share some features of known heterochromatic genes in their organization (MITCHELSON et al. 1993). Some DNA sequences within the region are underreplicated and virtually all of them are located within the chromocenter (MIKLOS and COTSELL 1990; YAMAMOTO et al. 1990; KORYAKOV et al. 1999). To visualize this region apart from the chromocenter and to dissect the distal Xh into fragments, we used a series of inversions with known breakpoints, which relocate different fragments of Xh next to the euchromatic sites. Since the PEV-inducing rearrangement In(1)wm4h (denoted hereafter as wm4) is usually used to monitor for the presence of Su(var)3-9 mutations (SCHOTTA et al. 2003; EBERT et al. 2004), we first examined a distinct and well-characterized block of distal Xh relocated to euchromatic region 3C1-2 in wm4.

We found that in the Su(var)3-9 mutant background the region 20A-F in the inverted X chromosome acquires a distinct banding pattern: in contrast to one or two bands usually detected in the region of either the wild-type or the wm4 X chromosome (KOLESNIKOVA et al. 2001; SEMESHIN et al. 2001), we managed to map up to 14–18 bands within this region (Figure 1, C and D). Furthermore, the chromosomal regions surrounding the eu–heterochromatin junction in the 20F-3C1-2 (DRYSDALE et al. 2005) region turned into an enormous "puff" (denoted hereafter as a pseudopuff, or PP) (Figure 1, C and D). Although varying in its extent from nucleus to nucleus, this impressive mutant phenotype is present in every wm4 X chromosome analyzed and is most prominent in males reared at 25° but not at 18°, thus appearing to be both temperature and sex sensitive. Similar mutant phenotypes were observed in Su(var)3-907/Su(var)3-906 and Su(var)3-922/Su(var)3-906 trans-heterozygotes (data not shown). Notably, the pseudopuff sizes are moderate in these mutants, probably because low levels of HMTase activity are still present due to mutant product expression and maternal effect provided by Su(var)3-9/TM6 mothers. The ectopic overexpression of Su(var)3-9 induced in the Sgs3-GAL4>UAS system in mid-third instar Su(var)3-906 larvae forces the chromocenter and inverted region 20 to recondense to different extents and the pseudopuff is strongly reduced (Figure 1E). Complete absence of the pseudopuff development was found in the control (Figure 1C). Thus, we conclude that it is the lack of wild-type level SU(VAR)3-9 HMTase activity that induces formation of a striking new pseudopuff and leads to the improved banding pattern in the proximal part of region 20. Additionally, we demonstrate that the pseudopuff develops in the Su(var)2-5 mutants lacking HP1 as well, although it is not as large (Figure 1F). Since recently it was shown that loss of SU(VAR)3-7 (one more constituent protein of heterochromatin) results in loosening of the chromocenter (SPIERER et al. 2005), we can suggest that the pseudopuff phenomenon could be observed in Su(var)3-7 mutants as well.

It should be noted that the SuUR mutation suppresses underreplication in IH and partially in PH regions and, therefore, it also "improves" the banding pattern of the 20 region to some extent (KOLESNIKOVA et al. 2001). To test possible genetic interactions of SuUR and Su(var)3-9 mutations, we combined wm4 with Su(var)3-9 and SuUR. In wm4; SuUR mutant females, region 20F-3C looks like a massive condensed chromosome block, larger than in the wm4 strain, probably because of suppression of underreplication of some heterochromatic sequences in the SuUR mutant (Figure 2A). A small but notable pseudopuff appears in wm4; Su(var)3-9 females (Figure 2B), while a tremendous pseudopuff develops in wm4; SuUR Su(var)3-9 females. SuUR and Su(var)3-906 appear to display additive effect in terms of improved structure of the entire pericentric region 20 in females (Figure 2C). Moreover, the most proximal part of region 20 looks decompacted even in female larvae from the y w, SuUR Su(var)3-9 strain carrying nonrearranged X chromosome, which implies that surprising pseudopuff is not inversion wm4 specific (Figure 2D).


Figure 2
View larger version (86K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 2.— Effect of SuUR mutation on the morphology of section 20 in wm4; Su(var)3-906 females. (A) A few distinct bands are observed in region 20 in wm4; SuUR females. Densely stained compacted block involves joined regions 20F and 3C. (B) Several new bands appear in region 20 and the pseudopuff is formed in wm4; Su(var)3-906 females. (C) In wm4; SuUR Su(var)3-906 females, the region 20A-F demonstrates a clear banding pattern, and the PP becomes enormous and occupies the region from 20F to 3B. (D) In yw; SuUR Su(var)3-906 females, improvement of the banding pattern and formation of the decompacted zone (PP) occur within nonrearranged region 20. Bar, 5 µm.

 
Pseudopuff develops from distal part of the X chromosome heterochromatin:
To identify the material participating in pseudopuff formation, we first used a set of inversions having breakpoints in the Xh and relocating its various parts to euchromatin (Figure 3J). This mapping was carried out for female SuUR Su(var)3-906 larvae, producing the nice chromosome morphology of region 20 (Figure 3, A–E). Data shown in Figure 3, A–C, demonstrate that inversions sc4, rst3, and wm4 break the pseudopuff as well and thus parts of the pseudopuff are seen on both sides of the heterochromatic breakpoints. At the same time, in the inversions scV2 and sc8 that break the X heterochromatin closer to the centromere, the pseudopuff arises only on one side of the inverted chromosome, namely on the distal (toward centromere) part of Xh (Figure 3, D and E). This indicates that heterochromatic blocks h30–h34 (Figure 3J) do not participate in pseudopuff formation.


Figure 3
View larger version (62K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 3.— Mapping of the pseudopuff within Xh by means of cytological and genetic markers in Su(UR) Su(var)3-906 mutant background. (A–E) Morphology of distal (XD) and proximal (XP) breakpoint regions of a series of inversions transferring different Xh blocks into the vicinity of euchromatic regions. White arrowheads point to a pseudopuff. Black arrowheads point to compacted proximal Xh regions. (F–I) FISH of the DNA markers in chromosomes with inversions: su(f) (F) and c23 (I) with wm4, hSte with sc4 (G), and 28S rDNA with rst3 (H). (J) Correspondence between mitotic X heterochromatic blocks and the polytene region 20 in SuUR Su(var)3-906 mutants. The scheme shows localization of inversion breakpoints and genetic loci within the Xh: (top) within the mitotic heterochromatin blocks and (bottom) within polytene region 20 in the SuUR Su(var)3-906 mutant background. The rst3 breakpoint is marked by a dashed line to denote its rough mapping within the h26–h28 blocks. The heterochromatic segments and rearrangement breakpoints are localized according to GATTI and PIMPINELLI (1992); bands in region 20 are according to KOLESNIKOVA et al. (2001). Bar, 5 µm.

 
For more correct identification of pseudopuff material, we performed FISH mapping using several DNA markers [su(f), hSte, 28S, and c23]. The su(f) gene, cytologically mapped at region 20E (SCHALET and LEFEVRE 1973; YAMAMOTO et al. 1990; KOLESNIKOVA et al. 2001), is considered to be a conditional "genetic boundary" between eu and heterochromatin of the X chromosome (SCHALET and LEFEVRE 1976). Data on FISH mapping of the su(f) DNA probe using the wm4 inversion indicate that the signal is located just before the pseudopuff and, therefore, the pseudopuff should be mapped next to 20E region, proximally to the region of decondensed bands that we conditionally call 20F1-4 (Figure 3, F and J; also see Figure 1D).

Two clusters of middle repetitive sequences are localized in distal Xh, hStellate and SCLR (Figure 3J) (NURMINSKY et al. 1994; TULIN et al. 1997). The Stellate genes mapped to the heterochromatic block h26 are tandemly repeated. Proximal to hSte (Figure 3J), SCLRs are composed of damaged heterochromatic variants of Stellate genes, copia-like elements, LINEs, and rDNA fragments. To map hSte and SCLR gene clusters, we used the hSte DNA fragment (common to both) as a probe for FISH with the sc4 inversion, which breaks the hSte repeats (TULIN et al. 1997). In accordance to this data, strong label signals are split and are seen on both sides of the inversion breakpoint in the region 20F1-4 and in the distal part of the pseudopuff (Figure 3G). Further, we used 28S rDNA for FISH with inversions rst3 (Figure 3H) and wm4 (data not shown). The 28S rDNA probe strongly labels two regions: the region corresponding to SCLR (the same localization that we determined for the hSte DNA probe) and the region located proximally to the pseudopuff [corresponding to rDNA genes, or the nucleolus organizer (NO)]. Additionally, c23 DNA, normally located in region 3C1-2, 2–3 kb distally from the wm4 breakpoint (TARTOF et al. 1984), labels pseudopuff and often looks diffused (Figure 3I), indicating that euchromatic region 3C of the X chromosome could sometimes be involved in pseudopuffing.

So, the data above demonstrate that the pseudopuff develops from chromosomal material corresponding to the proximal part of the h26–h28 mitotic heterochromatin blocks since (i) DNA clones located in the most distal part of heterochromatin of the mitotic X chromosome (h26) mark the most distal part of the pseudopuff and (ii) the NO (h29) proximally borders the pseudopuff but is not involved in it (Figure 3J).

The distribution of eu- and heterochromatin-specific proteins within the pseudopuff in the Su(var)3-9 mutants:
We next asked whether the alteration of compaction state in heterochromatin and pseudopuff formation in wm4; Su(var)3-906 nuclei would reflect changes in the localization pattern of heterochromatin-specific proteins. In a wild-type chromocenter, as well as in the wm4 X chromosome, all known heterochromatin-specific proteins completely colocalize and associate with the entire chromocenter (ZHIMULEV et al. 2004; DIMITRI et al. 2005). In the Su(var)3-906 background, HP1 is practically lost from the chromocenter except for weak binding to the core part and is unaffected at the fourth chromosome (SCHOTTA et al. 2002). We also found no notable HP1 signal in the pseudopuff and in both the XD and XP ends, but, unexpectedly, we often observed a weak binding site in the 20BC region and occasionally in 20F1-4 (Figure 4A). The same binding pattern was found for HP2, which has been described as an intimate partner of HP1, interacting with it both in vitro and in vivo (SHAFFER et al. 2002; STEPHENS et al. 2005) (Figure 4B).


Figure 4
View larger version (112K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 4.— Distribution of eu- and heterochromatin-specific proteins within region 20 of the wm4; Su(var)3-906 mutant X chromosome. (A–E) Localization of heterochromatin-specific proteins. (F–J) Localization of euchromatin-specific proteins. Brackets point to the H3Me3K4 site located within the distal border of an enormous pseudopuff; the site appears diffused. Merged images (phase contrast view/inverted immunofluorescent pattern) are shown. Proteins are indicated at the bottom of each panel. Bar, 5 µm.

 
H3 and H4 histones are generally hypoacetylated within PH (TURNER et al. 1992, SCHUEBELER et al. 2004). At the same time, acetylation of H4K12 is now considered to be required for heterochromatin formation and is upstream of H3K9 methylation (SWAMINATHAN et al. 2005). We observed the H4AcK12 pattern to be unaffected in a mutant chromocenter; however, pseudopuff staining appeared very weak or absent (Figure 4C). In contrast, two other heterochromatic proteins, SUUR and SU(VAR)3-7, bind intensively to both region 20BC and the entire body of the pseudopuff (Figure 4, D and E).

Next, we tested the distribution of several markers characteristic for active or decompacted chromatin (H3Me3K4, JIL-1, PolIIo, MSL2, and Z4) in wm4 control vs. wm4; Su(var)3-906 mutant X chromosomes. In the control, these proteins show similar localization patterns: they are associated with many interbands and/or puffs along the chromosome arms but are practically absent from the chromocenter (reviewed in ZHIMULEV et al. 2004) and from the compact heterochromatic block surrounding the wm4 breakpoint (data not shown). In wm4; Su(var)3-906 mutants, these proteins show strong binding to all corresponding euchromatic regions and, in particular, to interbands that become visible within the region 20A-E. Nevertheless, MSL2, H3Me3K4, JIL-1, and Z4 show common "gaps" both in region 20BC and in the pseudopuff, and PolIIo demonstrates weak binding along the entire region 20 (Figure 4, F–J).

So, decompaction of the heterochromatin region forming the pseudopuff is not accompanied by the appearance of the proteins characteristic of active chromosome regions; loss of the typical heterochromatin proteins SU(VAR)3-9, HP1, and HP2 does not prevent recruitment of SU(VAR)3-7. Unexpectedly, part of region 20BC is found to recruit all heterochromatin-specific proteins analyzed, thus serving as a small "island" of PH.

Loss of SU(VAR)3-9 affects replication status of the pseudopuff region:
We studied replication timing of the pseudopuff region by means of pulse incorporation of IdU. In wm4; SuUR chromosomes (no notable pseudopuff formation), the heterochromatic regions around the breakpoint of wm4 inversion are clearly late replicating (Figure 5, A and B). In relatively early stages of S-phase, the 20F-3C region does not incorporate the precursor (Figure 5A). When euchromatic regions have almost completed replication, region 20F-3C as well as compact zones within 20B-D are still replicating (Figure 5B). In both wm4; SuUR Su(var)3-906 and wm4; Su(var)3-906 mutants, the region of the pseudopuff is strongly labeled at relatively early stages of the S-period corresponding to the replication pattern shown on Figure 5D when numerous euchromatic regions incorporate the precursor, and the incorporation completes much earlier than in the neighboring heterochromatic regions 19E and 20C-F (Figure 5, C and E). Thus, the material of distal Xh, which undergoes decompaction in a Su(var)3-9 mutant background, also completes replication much earlier than it does as a compacted block.


Figure 5
View larger version (89K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 5.— Effect of mutations on IdU incorporation dynamics in distal heterochromatin. (A) Middle S-phase. Many bands are labeled, including 1B, 1C-E, 3A, 19A-F, and 20A-E. In wm4; SuUR mutants, the 20F-3C region forming the pseudopuff has not started replication. (B) Late S-phase. Only a few bands in intercalary and pericentric heterochromatin are labeled. In wm4; SuUR mutants, 20F-3C still incorporates the label. (C) The end of middle S-phase. A portion of the bands are intensively labeled, including 1B, 1E, 19E-F, and 20A-E. In wm4; Su(var)3-906 mutants, the pseudopuff has already completed replication. (D) Middle S-phase. In wm4; SuUR Su(var)3-906, the pseudopuff in the 20F-3C region incorporates DNA precursor. (E) Late S-phase. In wm4; SuUR Su(var)3-906, the pseudopuff has already completed the replication while 19E-20F regions are still labeled.

 
Since this change occurs in the pseudopuff region in mutants, we reasoned that at least some of the DNA sequences within the pseudopuff could be more polytenized than they were in the wild-type X chromosomes. To test this possibility, we studied relative abundance of the DNA fragments in eu and heterochromatin around the distal eu–heterochromatin junction of wm4 in males and females in both wild-type and SuUR and Su(var)3-9 backgrounds.

Several NotI restriction sites within the wild-type 3C region exist according to the Berkeley Drosophila Genome Project. Cutting at these sites generates a series of short fragments (0.4–7 kb) and two long fragments of 35 and 509 kb (Figure 6A). Presence of these fragments in DNA from different strains bearing nonrearranged X chromosomes of different origin was confirmed by Southern blot hybridization. In SuUR mutants (no underreplication in the IH region 3C), the DNA fragments of 509 and 35 kb are clearly seen (the 35-kb fragment is slightly weaker than the 509-kb one because of incomplete overlap with the probe; Figure 6B). In contrast, underreplication is particularly strong in 4xSuUR+ transgenic larvae (ZHIMULEV et al. 2003) and results in drastic weakening of the 509-kb signal. Moreover, we see a long smear of shorter fragments (240–400 kb) (Figure 6B). These sizes correspond very well to the distance between the NotI site and the site of underreplication in IH in this region (taken from BELYAKIN et al. 2005).


Figure 6
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 6.— Pulsed-field gel electrophoresis of DNA fragments located in the vicinity of the wm4 inversion breakpoint. Structures of nonrearranged white locus (A) and wm4 distal part (C) are shown. In C, the horizontal line represents euchromatic sequences in region 3C and the thick solid line represents sequences from the heterochromatic region. The restriction fragments analyzed in this report are shown as thin lines and their appropriate locations are given above the maps. The NotI (N) restriction sites that generate these fragments are shown. DNA fragment (c23) used as a probe for Southern blot hybridization is indicated by the shaded bar below the maps along with the Syx4 and white genes as reference points. Approximate position of truncation area within IH of 3C4-5 region is depicted as a dashed line. The coordinate system used for the wm4 distal part is indicated, where 0 kb is defined as the position of the wm4 distal rearrangement junction. Sequences to the right of the wm4 breakpoint extending into heterochromatic material are denoted by positive values and sequences to the left of the wm4 breakpoint are denoted by negative values (C). Results of Southern blot hybridization of the c23 probe with genomic DNA from the nonrearranged white locus (B) and wm4 distal part (D). Nuclei from salivary glands (SG) or imaginal discs-plus-brain complexes (ID+B) of larvae with indicated genotype and sex (m, male; f, female) were collected, DNA was isolated, digested with NotI, fractionated by pulsed-field gel electrophoresis, and analyzed by hybridization. The sizes of hybridized NotI fragments are shown by arrows near each panel. {lambda}-Phage DNA concatemers were used as a molecular mass ruler (M).

 
In the wm4 inversion, the X chromosome is broken between the w and Syx4 genes, the breakpoint locating 24.5 kb distally to w (TARTOF et al. 1984) (Figure 6C). Therefore, in this stock the 509-kb fragment is broken in two, and there is only 32 kb left from the euchromatic part, which is joined to the heterochromatic fragment now having a total length of ~75 kb (Figure 6C). Southern hybridization with c23 DNA shows that in the wm4 chromosome, in males and especially in females, intensity of the 75-kb signal is reduced notably in comparison to that of diploid cells DNA (Figure 6D). In contrast, intensity of signals for the 75-kb fragment in Su(var)3-906 mutants, SuUR mutants, and SuUR Su(var)3-906 double mutants is much higher and looks similar to that in diploid cells (Figure 6D). In all cases, hybridization intensities of the 75-kb fragment were compared to the intensity of the 35-kb euchromatic fragment (Figure 6D). So, absence of both SUUR and SU(VAR)3-9 proteins results in increase in the copy number of heterochromatic DNA fragments found on the distal eu–heterochromatin junction of wm4.

Surprisingly, we observed a reduction in the copy number of the 75-kb fragment concomitant with its underreplication rather than the smear of shortened fragments, similar to the pattern characteristic of underreplicated regions of intercalary heterochromatin (Figure 6D). We suggest that between the 75- and 35-kb fragments there is a region in genomic DNA that serves as a barrier for replication forks moving from the distal end of the X chromosome to the 3C breakpoint. However, this idea needs to be experimentally tested.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Recently it was shown that in Su(var)2-5 and Su(var)3-7 mutants the chromocenter in salivary gland nuclei looks relatively loose and diffuse (SPIERER et al. 2005). We found that loss or drastic reduction in the HMTase activity of SU(VAR)3-9 also results in strong decompaction of PH in polytene chromosomes. Most likely, only the functional complex of all these proteins can form compact pericentric heterochromatin.

We were interested in finding out to what degree the compact state of heterochromatin is dependent on specific heterochromatin proteins, particularly SU(VAR)3-9. Using a set of chromosome rearrangements placing different parts of X chromosome heterochromatin adjacent to euchromatin, we discovered that different portions of the polytene Xh are very differently affected by loss of SU(VAR)3-9: only the distal part of heterochromatin (heterochromatic blocks h26–h29 of the metaphase chromosome map according to GATTI and PIMPINELLI 1992) becomes decondensed to a varying extent, while morphology of the heterochromatic blocks h30–h34 appears unaffected.

The extent of heterochromatin decompaction depends on several different factors. The pseudopuff is more pronounced in males than in females. Although MSL2 protein, the core component of the dosage compensation complex (DCC), was not found in the pseudopuff (Figure 4I), we suggest that dosage compensation—a process equalizing X-linked gene expression in both sexes—might be responsible for this effect. It is known that DCC is distributed rather discretely, particularly, skipping over IH regions along the male X (ALEKSEYENKO et al. 2002). Nevertheless, it was shown that the whole X has a decondensed appearance and underreplication in IH regions is highly suppressed due to DCC function (ALEKSEYENKO et al. 2002). So, we can propose that in males the "puffed" portion of pericentric Xh relocated into the vicinity of euchromatin becomes dependent on dosage-compensating mechanisms, similarly to IH regions. Another possibility is that the Y chromosome, which represents an additional factor competing for the compaction proteins, might also contribute to the decompaction of Xh and to pseudopuff formation. To note, the fully heterochromatic Y chromosome comprises 40.9 Mb of DNA, while Xh contains ~20 Mb (ADAMS et al. 2000).

Among the inversions analyzed, wm4 produces the largest pseudopuff. A good explanation for this effect is currently missing; possibly, the chromatin environment in this eu–heterochromatin junction might contribute to Xh "puffing" or some of the DNA sequences might be differentially represented in Xh in different inversions. And finally, decondensation is most strongly expressed on the background of two mutations, Su(var)3-9 and SuUR, despite the fact that SuUR mutation itself does not induce "puffing" of Xh. The SuUR mutation results in additional polytenization of some of the Xh regions and thus it might increase the amount of Xh material capable of forming the pseudopuff. So, we believe that loss of both proteins, SUUR and SU(VAR)3-9, has an additive effect on the sizes of the decompacted region.

The region of decondensed heterochromatin that we call the pseudopuff does not demonstrate signs of true transcriptionally active puffs: the proteins characteristic for active decondensed chromatin (Z4, MSL2, JIL-1, and H3Me3K4) were not detected in the pseudopuff, with the exception of a very weak signal of PolIIo (Figure 4, F–J). At the same time, in the Su(var)3-9 mutants, HP1 and HP2 are weakly associated with the region 20F1-4, whereas SUUR and SU(VAR)3-7, in contrast, intensively bind the entire body of the pseudopuff (Figure 4, D and E). Recruitment of SU(VAR)3-7 into the pseudopuff in the absence of HP1 and SU(VAR)3-9 appears to be a very specific characteristic of pseudopuff heterochromatin since HP1 and SU(VAR)3-7 proteins were shown to cooperate closely in chromosome organization and development (SPIERER et al. 2005). Presence of the SUUR and SU(VAR)3-7 in decompacted chromatin of the pseudopuff might indicate that they do not participate in the process of compaction of this heterochromatic material or that they act in this direction only in the complex with functional SU(VAR)3-9.

It is interesting to note that different parts of Xh respond differentially to the removal of this complex. The question then, is which features of organization permit proximal heterochromatin to maintain its dense packing in the absence of HP1 and SU(VAR)3-9 activities? We might propose that these regions are under control of protein complexes of another composition. For example, these complexes might not utilize HMTase SU(VAR)3-9. However, data on position-effect variegation contradict this idea, since gene inactivation induced by chromosome rearrangements in proximal heterochromatin also depends on the SU(VAR)3-9 (KUHFITTIG et al. 2001). We could suggest that these complexes include some additional compacting proteins. It is known that, in contrast to the distal part of PH, its proximal part is enriched with satellite sequences that are associated with some specific proteins. For, example, the AT-hook protein D1 specifically binds to AT-rich satellites in deep Xh (AULNER et al. 2002).

In the course of investigating pseudopuff replication we found that underreplication of the heterochromatic sequences was suppressed in the region of the eu–heterochromatic junction of the wm4 inversion in Su(var)3-9 mutant. Thus, full polytenization of at least a 45-kb fragment adjacent to euchromatin occurs. The pseudopuff region, in general, completes replication before the end of S-phase; in other words, it does so earlier than the bulk of PH and even some IH regions. Still, a notable fraction of X chromosome heterochromatin sequences remains underreplicated in the polytene chromocenter. We can assume that some Xh regions not only do not complete replication but also do not start it (LILLI and SPRADLING 1996). Probably, these regions are separated from replicating chromatin by some barriers (LEACH et al. 2000) that prevent progression of replication forks from adjacent replicons. Therefore, for the first time we demonstrate that Su(var)3-906 may act as a suppressor of underreplication. However, we do not know how this mutation can affect underreplication in other heterochromatic regions.

Replication timing of the pseudopuff is notably changed in the absence of essential changes in transcriptional activity (the PolIIo painting of the pseudopuff looks no more intensive than that of the chromocenter). Moreover, the H3K4 methylation mark characteristic of active regions was not found in the pseudopuff at all. At the same time we can point to the correlation between the shift to earlier replication and chromatin decompaction. This observation is interesting in relation to cause–effect relationships among replication timing, transcriptional activity, and decompaction of chromatin (for review see DONALDSON 2005).

It is interesting to note that the effect of the SuUR mutation, known as suppression of underreplication (BELYAEVA et al. 1998), was found not only in PH but also in all IH regions and that these regions complete replication earlier in SuUR mutants than in wild-type ones (ZHIMULEV et al. 2003). The pseudopuff material in SuUR mutants is virtually at the same level of polytenization as in the Su(var)3-9 mutant. However, the SuUR mutation does not involve decompaction of the distal Xh. The same was noted for IH regions. Even if SuUR does induce decompaction of high-level chromatin structures, this is not detected by cytological means. Therefore, SuUR mutation affects replication timing differently compared to Su(var)3-9.

Summing up, we can conclude that the reaction of pericentric heterochromatin to loss of SU(VAR)3-9 and HP1 varies in different regions of X heterochromatin. Only the distal part of it undergoes decondensation and forms a new structure called a pseudopuff, which has a specific organization, demonstrating some characteristics of active chromatin: decompaction and, concomitantly, earlier completion of replication. At the same time, the pseudopuff does not contain proteins of active chromatin but does contain several heterochromatic proteins.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We are grateful to S. Elgin for HP1 and HP2 antibodies, A. Spierer and P. Spierer for SU(VAR)3-7 antibodies, H. Saumweber for Z4 antibodies, J. Johansen for JIL-1 antibodies, and M. Kuroda for MSL2 antibodies. We also thank G. Reuter for fly strains Su(var)3-907, Su(var)3-906, Su(var)3-922, and UAS-Su(var)3-9-EGFP; V. Pirrotta for cosmid c23 DNA; K. O'Hare for su(f) DNA; N. Tchurikov for 28S rDNA; and V. Gvozdev for hSte DNA. We thank L. Boldyreva for creating the SuUR Su(var)3-9 strain and P. Spierer and A. Gortchakov for reading and commenting on the manuscript. The work was supported by grants from Molecular and Cellular Biology Russian Academy of Sciences program N10.1, Interdisciplinary Integration Project of Siberian Branch of Russian Academy of Sciences N45, grant from Russian Foundation for Basic Researches 06-04-48387-a.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

ADAMS, M. D., S. E. CELNIKER, R. A. HOLT, C. A. EVANS, J. D. GOCAYNE et al., 2000 The genome sequence of Drosophila melanogaster. Science 287: 2185–2195.[Abstract/Free Full Text]

AHMAD, K., and S. HENIKOFF, 2002 Histone H3 variants specify modes of chromatin assembly. Proc. Natl. Acad. Sci. USA 99: 16477–16484.[Abstract/Free Full Text]

ALEKSEYENKO, A. A., O. V. DEMAKOVA, E. S. BELYAEVA, G. F. MAKAREVICH, I. V. KOTLIKOVA et al., 2002 Dosage compensation and intercalary heterochromatin in X chromosomes of Drosophila melanogaster. Chromosoma 111: 106–113.[Medline]

ANDREYEVA, E. N., E. S. BELYAEVA, V. F. SEMESHIN, G. V. POKHOLKOVA and I. F. ZHIMULEV, 2005 Three distinct chromatin domains in telomere ends of polytene chromosomes in Drosophila melanogaster Tel mutants. J. Cell Sci. 118: 5465–5477.[Abstract/Free Full Text]

AULNER, N., C. MONOD, G. MANDICOURT, D. JULLIEN, O. CUVIER et al., 2002 The AT-hook protein D1 is essential for Drosophila melanogaster development and is implicated in position-effect variegation. Mol. Cell. Biol. 22: 1218–1232.[Abstract/Free Full Text]

BANNISTER, A. J., P. ZEGERMAN, J. F. PARTRIDGE, E. A. MISKA, J. O. THOMAS et al., 2001 Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120–124.[CrossRef][Medline]

BELYAEVA, E. S., O. V. DEMAKOVA, G. H. UMBETOVA and I. F. ZHIMULEV, 1993 Cytogenetic and molecular aspects of position-effect variegation in Drosophila melanogaster. V. Heterochromatin-associated protein HP1 appears in euchromatic chromosomal regions that are inactivated as a result of position-effect variegation. Chromosoma 102: 583–590.[CrossRef][Medline]

BELYAEVA, E. S., I. F. ZHIMULEV, E. I. VOLKOVA, A. A. ALEKSEYENKO, Y. M. MOSHKIN et al., 1998 Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes. Proc. Natl. Acad. Sci. USA 95: 7532–7537.[Abstract/Free Full Text]

BELYAEVA, E. S., L. V. BOLDYREVA, E. I. VOLKOVA, R. A. NANAYEV, A. A. ALEKSEYENKO et al., 2003 Effect of the Suppressor of Underreplication (SuUR) gene on position-effect variegation silencing in Drosophila melanogaster. Genetics 165: 1209–1220.[Abstract/Free Full Text]

BELYAKIN, S. N., G. K. CHRISTOPHIDES, A. A. ALEKSEYENKO, E. V. KRIVENTSEVA, E. S. BELYAEVA et al., 2005 Genomic analysis of Drosophila chromosome underreplication reveals a link between replication control and transcriptional territories. Proc. Natl. Acad. Sci. USA 102: 8269–8274.[Abstract/Free Full Text]

BERGHELLA, L., and P. DIMITRI, 1996 The heterochromatic rolled gene of Drosophila melanogaster is extensively polytenized and transcriptionally active in the salivary gland chromocenter. Genetics 144: 117–125.[Abstract]

BRAND, A. H., and N. PERRIMON, 1993 Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.[Abstract]

CHERBAS, L., X. HU, I. ZHIMULEV, E. BELYAEVA and P. CHERBAS, 2003 EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue. Development 130: 271–284.[Abstract/Free Full Text]

CLEARD, F., and P. SPIERER, 2001 Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein. EMBO J. 2: 1095–1100.[CrossRef]

CLEARD, F., M. DELATTRE and P. SPIERER, 1997 SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation. EMBO J. 16: 5280–5288.[CrossRef][Medline]

CZERMIN, B., R. MELFI, D. MCCABE, V. SEITZ, A. IMHOF et al., 2002 Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111: 185–196.[CrossRef][Medline]

DELATTRE, M., A. SPIERER, C.-H. TONKA and P. SPIERER, 2000 The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. J. Cell Sci. 113: 4253–4261.[Abstract]

DELATTRE, M., A. SPIERER, Y. JAQUET and P. SPIERER, 2004 Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J. Cell Sci. 117: 6239–6247.[Abstract/Free Full Text]

DEVLIN, R. H., B. BINGHAM and B. T. WAKIMOTO, 1990 The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster. Genetics 125: 129–140.[Abstract]

DIMITRI, P., N. CORRADINI, F. ROSSI and F. VERNI, 2005 The paradox of functional heterochromatin. BioEssays 27: 29–41.[CrossRef][Medline]

DONALDSON, A. D., 2005 Shaping time: chromatin structure and the DNA replication programme. Trends Genet. 21: 444–449.[CrossRef][Medline]

DRYSDALE, R. A., M. A. CROSBY and FLYBASE CONSORTIUM, 2005 FlyBase: genes and gene models. Nucleic Acids Res. 33: D390–D395 (http://flybase.org).[Abstract/Free Full Text]

EBERT, A., G. SCHOTTA, S. LEIN, S. KUBICEK, V. KRAUSS et al., 2004 Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev. 18: 2973–2983.[Abstract/Free Full Text]

EISSENBERG, J. C., and S. ELGIN, 2000 The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev. 10: 204–210.[CrossRef][Medline]

ELGIN, S. C., 1996 Heterochromatin and gene regulation in Drosophila. Curr. Opin. Genet. Dev. 6: 193–202.[CrossRef][Medline]

GATTI, M., and S. PIMPINELLI, 1992 Functional elements in Drosophila melanogaster heterochromatin. Annu. Rev. Genet. 26: 239–275.[CrossRef][Medline]

GATTI, M., S. BONACCORSI and S. PIMPINELLI, 1994 Looking at Drosophila mitotic chromosomes. Methods Cell Biol. 44: 371–391.[Medline]

GREIL, F., I. VAN DER KRAAN, J. DELROW, J. F. SMOTHERS, E. DE WIT et al., 2003 Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17: 2825–2838.[Abstract/Free Full Text]

GREWAL, S. I., and S. C. ELGIN, 2002 Heterochromatin: new possibilities for the inheritance of structure. Curr. Opin. Genet. Dev. 12: 178–187.[CrossRef][Medline]

KAMAKAKA, R. T., and S. BIGGINS, 2005 Histone variants: Deviants? Genes Dev. 19: 295–310.[Abstract/Free Full Text]

KARPEN, G. H., and A. C. SPRADLING, 1990 Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila. Cell 63: 97–107.[CrossRef][Medline]

KOLESNIKOVA, T. D., D. E. KORIAKOV, V. F. SEMESHIN, E. S. BELIAEVA and I. F. ZHIMULEV, 2001 Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands. Genetika 37: 1632–1641 (in Russian).[Medline]

KORYAKOV, D. E., A. A. ALEKSEYENKO and I. F. ZHIMULEV, 1999 Dynamic organization of the ß-heterochromatin in the Drosophila melanogaster polytene X chromosome. Mol. Gen. Genet. 260: 503–509.[CrossRef][Medline]

KORYAKOV, D. E., E. V. DOMANITSKAYA, S. N. BELYAKIN and I. F. ZHIMULEV, 2003 Abnormal tissue-dependent polytenization of a block of chromosome 3 pericentric heterochromatin in Drosophila melanogaster. J. Cell Sci. 116: 1035–1044.[Abstract/Free Full Text]

KUHFITTIG, S, J. SZABAD, G. SCHOTTA, J. HOFFMANN, E. MATHE et al., 2001 pitkin(D), a novel gain-of-function enhancer of position-effect variegation, affects chromatin regulation during oogenesis and early embryogenesis in Drosophila. Genetics 157: 1227–1244.[Abstract/Free Full Text]

LACHNER, M., D. O'CARROLL, S. REA, K. MECHTLER and T. JENUWEIN, 2001 Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: 116–120.[CrossRef][Medline]

LE, M.-H., D. DURICKA and G. H. KARPEN, 1995 Islands of complex DNA are widespread in Drosophila centric heterochromatin. Genetics 141: 283–303.[Abstract]

LEACH, T. J., H. L. CHOTKOWSKI, M. G. WOTRING, R. L. DILWITH and R. L. GLASER, 2000 Replication of heterochromatin and structure of polytene chromosomes. Mol. Cell. Biol. 20: 6308–6316.[Abstract/Free Full Text]

LI, Y., D. A. KIRSCHMANN and L. L. WALLRATH, 2002 Does heterochromatin protein 1 always follow code? Proc. Natl. Acad. Sci. USA 99: 16462–16469.[Abstract/Free Full Text]

LILLY, M. A., and A. C. SPRADLING, 1996 The Drosophila endocycle is controlled by cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 10: 2514–2526.[Abstract/Free Full Text]

MIKLOS, G. L. G., and J. N. COTSELL, 1990 Chromosome structure at interfaces between major chromatin types: {alpha} and ß-heterochromatin. BioEssays 12: 1–9.[CrossRef][Medline]

MITCHELSON, A., M. SIMONELIG, C. WILLIAMS and K. O'HARE, 1993 Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability. Genes Dev. 7: 241–249.[Abstract/Free Full Text]

MOSHKIN, Y. M., S. N. BELYAKIN, N. B. RUBTSOV, E. B. KOKOZA, A. A. ALEKSEYENKO et al., 2002 Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogaster mutant Suppressor of Underreplication. Chromosoma 111: 114–125.[Medline]

NAKAYAMA, J., J. C. RICE, B. D. STRAHL, C. D. ALLIS and S. I. GREWAL, 2001 Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292: 110–113.[Abstract/Free Full Text]

NURMINSKY, D. I., Y. YA. SHEVELYOV, S. V. NUZHDIN and V. A. GVOZDEV, 1994 Structure, molecular evolution and maintenance of copy number of extended repeated structures in the X-heterochromatin of Drosophila melanogaster. Chromosoma 103: 277–285.[Medline]

PERRINI, B., L. PIACENTINI, L. FANTI, F. ALTIERI, S. CHICHIARELLI et al., 2004 HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila. Mol. Cell 15: 467–476.[CrossRef][Medline]

PIRROTTA, V., C. HADFIELD and G. H. PRETORIUS, 1983 Microdissection and cloning of the white locus and the 3B1–3C2 region of the Drosophila X chromosome. EMBO J. 2: 927–934.[Medline]

REA, S., F. EISENHABER, D. O'CARROLL, B. D. STRAHL, Z. W. SUN et al., 2000 Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593–599.[CrossRef][Medline]

RICHARDS, E. J., and S. C. ELGIN, 2002 Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108: 489–500.[CrossRef][Medline]

SCHALET, A., and G. LEFEVRE, 1973 The localization of ‘ordinary’ sex-linked genes in section 20 of the polytene chromosome of Drosophila melanogaster. Chromosoma 44: 183–202.[CrossRef][Medline]

SCHALET, A., and G. LEFEVRE, JR., 1976 The proximal region of the X chromosome, pp. 848–902 in The Genetics and Biology of Drosophila, edited by M. ASHBURNER and E. NOVITSK. Academic Press, London/New York/San Francisco.

SCHOTTA, G., A. EBERT, V. KRAUSS, A. FISCHER, J. HOFFMANN et al., 2002 Central role of Drosophila SU(VAR)3-9 in histone H3–K9 methylation and heterochromatic gene silencing. EMBO J. 21: 1121–1131.[CrossRef][Medline]

SCHOTTA, G, A. EBERT and G. REUTER, 2003 SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing. Genetica 117: 149–158.[CrossRef][Medline]

SCHUBELER, D., D. M. MACALPINE, D. SCALZO, C. WIRBELAUER, C. KOOPERBERG et al., 2004 The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18: 1263–1271.[Abstract/Free Full Text]

SEMESHIN, V. F., E. S. BELYAEVA and I. F. ZHIMULEV, 2001 Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster. Chromosoma 110: 487–500.[CrossRef][Medline]

SHAFFER, C. D., G. E. STEPHENS, B. A. THOMPSON, L. FUNCHES, J. A. BERNAT et al., 2002 Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin. Proc. Natl. Acad. Sci. USA 99: 14332–14337.[Abstract/Free Full Text]

STEPHENS, G. E., E. E. SLAWSON, C. A. CRAIG and S. C. ELGIN, 2005 Interaction of heterochromatin protein 2 with HP1 defines a novel HP1-binding domain. Biochemistry 44: 13394–13403.[CrossRef][Medline]

SPIERER, A., C. SEUM, M. DELATTRE and P. SPIERER, 2005 Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation. J. Cell Sci. 118: 5047–5057.[Abstract/Free Full Text]

SUN, X., H. D. LE, J. M. WAHLSTROM and G. H. KARPEN, 2003 Sequence analysis of a functional Drosophila centromere. Genome Res. 13: 182–194.[Abstract/Free Full Text]

SWAMINATHAN, J., E. M. BAXTER and V. G. CORCES, 2005 The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes. Dev. 19: 65–76.[Abstract/Free Full Text]

TARTOF, K. D., C. HOBBS and M. JONES, 1984 A structural basis for variegating position effects. Cell 37: 869–878.[CrossRef][Medline]

TAUTZ, D., J. M. HANCOCK, D. A. WEBB, C. TAUTZ and G. A. DOVER, 1988 Complete sequences of the rRNA genes of Drosophila melanogaster. Mol. Biol. Evol. 5: 366–376.[Abstract]

TSCHIERSCH, B., A. HOFMANN, V. KRAUSS, R. DORN, G. KORGE et al., 1994 The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13: 3822–3831.[Medline]

TULIN, A. V., G. L. KOGAN, D. FILIPP, M. D. BALAKIREVA and V. A. GVOZDEV, 1997 Heterochromatic Stellate gene cluster in Drosophila melanogaster: structure and molecular evolution. Genetics 146: 253–262.[Abstract]

TURNER, B. M., A. J. BIRLEY and J. LAVENDER, 1992 Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69: 375–384.[CrossRef][Medline]

WEILER, K. S., and B. T. WAKIMOTO, 1995 Heterochromatin and gene expression in Drosophila. Annu. Rev. Genet. 29: 577–605.[CrossRef][Medline]

YAMAMOTO, M. T., A. MITCHELSON, M. TUDOR, K. O'HARE, J. A. DAVIES et al., 1990 Molecular and cytogenetic analysis of the heterochromatin-euchromatin junction region of the Drosophila melanogaster X chromosome using cloned DNA sequences. Genetics 125: 821–832.[Abstract]

ZHIMULEV, I. F., 1998 Polytene chromosomes, heterochromatin and position effect variegation. Adv. Genet. 37: 1–566.[Medline]

ZHIMULEV, I. F., and E. S. BELYAEVA, 2003 Intercalary heterochromatin and genetic silencing. BioEssays 25: 1040–1051.[CrossRef][Medline]

ZHIMULEV, I. F., E. S. BELYAEVA, I. V. MAKUNIN, V. PIRROTTA, E. I. VOLKOVA et al., 2003 Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes. Chromosoma 111: 377–398.[Medline]

ZHIMULEV, I. F., E. S. BELYAEVA, V. F. SEMESHIN, D. E. KORYAKOV, S. A. DEMAKOV et al., 2004 Polytene chromosomes: 70 years of genetic research. Int. Rev. Cytol. 241: 203–275.[Medline]

Communicating editor: K. G. GOLIC




This article has been cited by other articles: