Department of Zoology and Animal Biology, University of Geneva, 30, quai Ernest-Ansermet, 1211, Geneva 4, Switzerland
* Author for correspondence (e-mail: pierre.spierer{at}zoo.unige.ch)
Accepted 21 September 2004
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Summary |
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Key words: Drosophila, Su(var)3-7, Heterochromatin, Position-effect variegation
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Introduction |
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Su(var)3-7 is a third modifier of PEV, which appears to be a structural component of heterochromatin. Su(var)3-7 encodes a protein mainly associated with pericentromeric heterochromatin and telomeres (Cléard et al., 1997; Delattre et al., 2000
). It encodes a large protein of 1169 amino acids that contains seven atypical and widely spaced zinc finger motifs, which were shown to bind DNA in vitro (Reuter et al., 1990
; Cléard et al., 1995
; Cléard and Spierer, 2001
). Specific binding to pericentric heterochromatin is conferred to Su(var)3-7 by its C-terminal region (Jaquet et al., 2002
). Extra doses of Su(var)3-7 enhance silencing of variegating genes, whereas a decreased level of Su(var)3-7 reduces the silencing (Reuter et al., 1990
). Loss of the Su(var)3-7 protein results in lethality, making it an essential protein for the fly. Interestingly, males are more sensitive to the lack of Su(var)3-7 than females, but the cause of lethality is unknown (Seum et al., 2002
). Although the Su(var)3-7 gene was identified more than ten years ago as a modifier of PEV, the exact role of its protein in heterochromatin structure is still poorly understood.
There are links between Su(var)3-7 and HP1. First the two genes show strong genetic interaction (Cléard et al., 1997). Second, the two proteins colocalise in the Drosophila embryos and on polytene chromosomes. Third, physical interaction between HP1 and Su(var)3-7 has been demonstrated by coimmunoprecipitation and yeast two-hybrid analysis (Cléard et al., 1997
; Delattre et al., 2000
). Su(var)3-7 was also shown to interact by two-hybrid interaction trapping in yeast with the histone methyltransferase Su(var)3-9 (Schotta et al., 2002
), but this interaction has not been confirmed in vivo until now. Despite these indications, the mode of action of Su(var)3-7 remains elusive. To address the role of Su(var)3-7, we analysed in vivo the consequences of increased Su(var)3-7 expression on fly viability and chromatin structure.
Here, we provide evidence for a dose-dependent regulatory role of Su(var)3-7 in chromosome morphology and heterochromatin formation and demonstrate in vivo a functional link between Su(var)3-7 and the histone methyltransferase Su(var)3-9.
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Materials and Methods |
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Immunostaining of polytene chromosomes
Procedures for immunostaining were as described (Platero et al., 1995). Briefly, salivary glands were dissected in Cohen's buffer, fixed for 2 minutes in 2% formaldehyde, 2% Triton X-100 and then squashed in 2% formaldehyde, 45% acetic acid. These fixation conditions were used for all immunostaining except in one case, as indicated in the text, where milder fixation consisted of squashing directly in 2% formaldehyde, 45% acetic acid. Primary antibodies were used at the following dilutions: 1:10 for anti-Su(var)3-7 (Cléard et al., 1997
), 1:20 for anti-H3-diMeK9 from Upstate, 1:400 for anti-HP1 (a gift of Lori Wallrath), 1:200 for anti-GAGA (a gift of Vincenzo Pirrotta) and 1:100 for anti-H3-diMeK27 (a gift of Thomas Jenuwein).
Orcein staining of polytene chromosomes
Larvae were dissected, and salivary glands transferred and squashed in 45% acetic acid. Slides were dehydrated for at least 20 minutes in 100% ethanol and air-dried. A drop of staining solution (1% orcein in a 1:1 mix of 60% acetic acid and lactic acid) was deposited on a coverslip and applied on the polytene chromosomes. Excess staining solution was removed and the coverslip sealed with nail varnish.
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Results |
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Chromosome morphology, particularly that of the male X, is profoundly affected by the dose of Su(var)3-7
As Su(var)3-7 is a chromosome-associated protein, we examined chromosome morphology in larvae expressing high level of the protein. Polytene chromosomes from HA:FL4D third instar larvae submitted to daily heat-shocks from the second instar larval stage were squashed and stained with orcein. High levels of Su(var)3-7 induce dramatic changes in chromosome morphology. First, in males as in females the chromocentre reproducibly appears denser when compared to non heat-shocked controls (Fig. 1A). Second, the morphology of the chromosome arms is affected: the banding pattern is altered, and more strikingly the male X chromosome becomes very small and compact (Fig. 1B). In this very spectacular phenotype, the length of the male X chromosome can be reduced by more than tenfold by increasing the expression of just one protein. Third, with stronger heat-shocks, starting from the first instar larval stage, extreme phenotypes appear (Fig. 1B): in males all the chromosomes are reduced in size, with the X always the most affected, and in females chromosomes also start to condense. These defects are never observed on non heat-shocked chromosomes, and are never observed on the control yw67 line submitted to the same treatment. These phenotypes are not specific to the HA:FL4D line: the HA:FL1A line, which contains the same full-length Su(var)3-7 transgene but inserted at a different location, shows the same effects although to a lesser extent, probably due to a lower level of induction (not shown). Fourth, the male X chromosome exhibits a reproducible novel array of bands and interbands. Compaction of the chromosome is not uniform and we surmise that some regions are specifically condensed while others are not. Taken together these observations provide evidence for a dose-dependent regulatory role of Su(var)3-7 in chromosome morphology.
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Su(var)3-7 invades chromosome euchromatic arms, particularly the male X, and induces methylation of H3-K9
We next assessed the consequences of excess of Su(var)3-7 on its association with chromosomes. Immunostaining of wild-type polytene chromosomes with anti-Su(var)3-7 antibodies shows staining restricted to the chromocentre and some telomeres (Delattre et al., 2000) (Fig. 2A). Staining for Su(var)3-7 in HA:FL4D male and female larvae submitted to daily heat-shocks reveals however that the protein invades all the chromosomes extensively (Delattre et al., 2000
) (Fig. 2A). Interestingly, in milder conditions of fixation, Su(var)3-7 is seen only on the chromocentre and on the compacted male X chromosome, probably revealing a stronger binding or a better affinity to these regions (Fig. 2B).
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We propose that the reduction in size and the alteration of the banding pattern of the chromosomes, especially of the male X chromosome, is due to heterochromatinisation. To test this hypothesis, we stained the HA:FL4D chromosomes with an antibody raised against H3-diMeK9, a marker of heterochromatin. Without heat-shock induction of Su(var)3-7, this modification of histone H3 is indeed detected mainly at the chromocentre and on a few telomeres, as already described (Jacobs et al., 2001; Li et al., 2002
) (Fig. 3). However, after heat-shock induction of Su(var)3-7, H3-diMeK9 is detected on many sites of all chromosomes in males and in females. Moreover, the male X chromosome is specifically almost entirely covered by the H3-diMeK9 staining (Fig. 3). The same strong enhancement of H3-diMeK9 staining is visible on the HA:FL1A male X chromosome even if the X is not greatly reduced in size in this line (Fig. 3).
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We investigated this particular sensitivity of the male X chromosome to overproduction of Su(var)3-7. First, we verified that this effect was not specific to the X chromosome of the yw67 fly stock containing the HA:FL construct by exchanging this X chromosome with the X from Canton S or w1118 stocks. Hypermethylation of H3-diMeK9 after overexpression of Su(var)3-7 was also observed on these two X chromosomes (not shown). We also verified that the increase of the H3-diMeK9 staining was not due to the change in the X morphology (as the chromosome becomes more compact, the staining of discrete bands scattered on the chromosome could appear continuous). We stained the chromosomes of the HA:FL4D line with antibodies against another modified histone, H3-diMeK27 (dimethylated on lysine 27) and with antibodies against the chromatin protein GAGA. Excess Su(var)3-7 did not increase the pattern of these antibodies specifically on the compacted X chromosome, although the general aspect of the staining is slightly modified by the change of morphology of all the chromosomes (Fig. 4 and data not shown). In conclusion, an increase in dose of Su(var)3-7 induces a heterochromatin-specific modification of histone H3 all over the chromosomes and most dramatically on the male X chromosome.
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Accumulation of Su(var)3-7 on male X chromosome induces recruitment of the HP1 protein
Knowing first that HP1 and Su(var)3-7 interact (Cléard et al., 1997; Delattre et al., 2000
), second that HP1 specifically binds H3-diMeK9 (Bannister et al., 2001
; Lachner et al., 2001
) and third that HP1 plays a crucial role in heterochromatin formation and gene silencing (Ayyanathan et al., 2003
; Li et al., 2003
), we tested whether excess Su(var)3-7 modifies the localisation of HP1. Polytene chromosomes of HA:FL4D larvae were stained with the anti-HP1 antibody (Fig. 5). Without heat-shock induction of Su(var)3-7, HP1 associates preferentially with pericentric heterochromatin, where it colocalises with H3-diMeK9 (Fig. 5). However, with an excess of Su(var)3-7, HP1 strongly decorates the entire male X chromosome in addition to the chromocentre (Fig. 5). On autosomes, overproduction of Su(var)3-7 increases the number of sites bound by HP1 (not shown). The recruitment of HP1 on the entire male X chromosome, together with the condensation and the increase of amount of H3-diMeK9, constitutes the third mark of heterochromatin formation induced by high levels of Su(var)3-7.
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Increased methylation of H3-K9 depends on Su(var)3-9
At least four different histone methyl-transferases selectively methylate H3 at lysine 9 (Rea et al., 2000; Tachibana et al., 2002
; Schultz et al., 2002
; Ogawa et al., 2002
). In contrast with the three others, Su(var)3-9 and its orthologues function primarily in heterochromatin. Moreover, Su(var)3-9 interacts in vivo with HP1, and interacts in the yeast two-hybrid system with Su(var)3-7 (Schotta et al., 2002
). We therefore tested the requirement for Su(var)3-9 on the increased methylation of H3-K9 induced by Su(var)3-7. We constructed a line harbouring the HA:FL1A transgene and the Su(var)3-906 null mutation (Tschiersch et al., 1994
), both of which are homozygous. We submitted larvae from this genotype to daily heat-shocks and examined the resulting chromosome morphology and H3-diMeK9 pattern. As a control, we analysed separate HA:FL1A and Su(var)3-906 genotypes. First, we observed that the morphology of the male X chromosome of HA:FL1A;Su(var)3-906 larvae is not altered by a high dose of Su(var)3-7 whereas that of HA:FL1A is affected (Fig. 6 and orcein staining not shown). Second, the pattern of H3-diMeK9 in HA:FL1A;Su(var)3-906 larvae is very similar to the Su(var)3-906 control but dramatically different from the HA:FL1A larvae (Fig. 6A). H3-diMeK9 staining does not accumulate on the X chromosome and is strongly reduced on euchromatic arms and on the chromocentre, except for a reproducible bright point (Fig. 6A) (Schotta et al., 2002
). In the same conditions HA:FL1A control chromosomes show strong accumulation of staining on the male X chromosome and over autosomes (not shown). This suggests that Su(var)3-9 is responsible not only for the methylation of H3-K9 at the chromocentre as previously reported (Schotta et al., 2002
), but also for the hypermethylation of the male X chromosome and autosomes triggered by increasing expression of Su(var)3-7. Interestingly, HP1 binding is also dramatically different in HA:FL1A; Su(var)3-906 larvae when compared with HA:FL1A larvae. HP1 staining is indeed strongly reduced everywhere, except for a reproducible bright point at the chromocentre (Fig. 6B). We conclude that the recruitment of HP1 by overproduced Su(var)3-7 requires functional Su(var)3-9.
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The loss of histone modification observed in a Su(var)3-906 mutant background could nevertheless result from a loss of expansion of Su(var)3-7 on these chromosomes. To test this possibility, we examined whether Su(var)3-7 binding on chromosomes was altered by the loss of Su(var)3-9. Immunostaining clearly showed that it is not the case: Su(var)3-7 is still bound to all chromosomes, in euchromatin as in heterochromatin, without the influence of Su(var)3-9 (Fig. 6B). The ability of Su(var)3-7 to spread on chromosomes does not depend on Su(var)3-9. Interestingly, we observed that the combination of HA:FL1A with Su(var)3-906 partially rescues the lethality induced by the overexpression of Su(var)3-7. Whereas all HA:FL1A individuals die after heat-shock, about 10% of HA:FL1A;Su(var)3-906 flies survive to adulthood without obvious phenotype (not shown). In conclusion, these results demonstrate that Su(var)3-7 and Su(var)3-9 are partners in vivo and that effects induced by overproduced Su(var)3-7 require wild-type amounts of Su(var)3-9.
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Discussion |
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Given that an increase in the dose of Su(var)3-7 provokes ectopic heterochromatinisation, we would expect an opposite phenotype in the absence of Su(var)3-7. This is indeed the case: an extensive analysis of previously described Su(var)3-7 mutations (Seum et al., 2002) revealed that loss of the protein results in decondensation of the male X chromosome (A.S., M.D., C.S. and P.S., in preparation). In addition, we have noticed that males suffer more than females from the lack of Su(var)3-7: the rescue of the lethality of Su(var)3-7 mutations by Su(var)3-7 transgenes was better in females than in males (Seum et al., 2002
). This observation is consistent with a particular susceptibility of the male X chromosome to the lack of Su(var)3-7. Decreased and increased levels of Su(var)3-7 have opposite phenotypes on the male X chromosome, showing that the effects described here are specific and that Su(var)3-7 plays a particular role, directly or indirectly, on the morphology of the male X chromosome.
The reason for the particular susceptibility of the male X chromosome in response to change of Su(var)3-7 dose is not yet clear. The main difference between the male X and other chromosomes is its interaction with the dosage compensation complex. In Drosophila, equalisation of X-linked gene expression between males and females is accomplished by a twofold hyper-transcription of most genes on the male X chromosome (reviewed by Akhtar et al., 2003). A complex made of male-specific proteins and RNAs binds to the male X and modifies histone tails. This unique chromatin environment may render the male X chromosome more accessible to Su(var)3-7, or increase the affinity of the protein to it. We are currently exploring the possibility of an interaction between Su(var)3-7 and the dosage compensation complex.
Spreading of Su(var)3-7 on euchromatin depends on its expression level
The increase of Su(var)3-7 levels reveals a new dimension to the previously described specificity of Su(var)3-7 binding. The protein is detected not only at the chromocentre but also on euchromatic arms in males as in females. These data suggest that in wild-type conditions, the heterochromatin restricted binding of Su(var)3-7 needs a precise control of the level of its expression. The dose-dependent capacity of Su(var)3-7 to spread all over chromosomes is reminiscent of the dose-dependent effect of Su(var)3-7 on PEV: the loss of a gene results in a strong suppression of variegation, whereas extra doses of the gene progressively enhance silencing of genes in the vicinity of heterochromatin (Reuter et al., 1990). The capacity of Su(var)3-7 to invade euchromatin might result from its general affinity for DNA (Cléard and Spierer, 2001
). When in excess, and without excesses of its heterochromatic partners like HP1, Su(var)3-7 could escape the heterochromatic complex and invade the rest of the chromosome only because of its affinity for DNA. This invasion property is not purely random as Su(var)3-7 shows preferential association with the male X chromosome. The non-monotonous compaction of euchromatin also reveals that Su(var)3-7 finds sites of higher affinity on euchromatic arms, and we suggest that it could expand from these sites as it does in position-effect variegation.
Su(var)3-7 and Su(var)3-9 interact in vivo
We first observed that an excess of Su(var)3-7 increases the number of sites on the chromosomes containing the H3-diMeK9, especially on the male X chromosome. Then we provided genetic evidence that the histone methyl-transferase leading to this increase is Su(var)3-9. In addition, we determined that the lethality induced by high levels of Su(var)3-7 is partially dependent on wild-type dose of Su(var)3-9. This is consistent with the fact that the suppressor effect of Su(var)3-9 loss of function is epistatic to the triploenhancer effect of Su(var)3-7 on PEV (Schotta et al., 2002). These pieces of evidence suggest that the function of Su(var)3-7 in silencing requires the wild-type activity of Su(var)3-9. Su(var)3-7 is able to expand the site of action of the histone H3 methyl-transferase, normally mainly restricted to the chromocentre. Conversely, expansion of Su(var)3-7 binding is not dependent on Su(var)3-9. Our study shows for the first time, evidence that Su(var)3-7 and Su(var)3-9 truly interact in vivo. This validates the interaction observed between the two proteins in the yeast two-hybrid system (Schotta et al., 2002
). Although Su(var)3-7 produced in excess is able to cover all chromosomes, methylation of H3-K9 occurs only at a number of loci on autosomes and almost uniformly on the male X chromosome. We conclude that ectopic methylation of H3K9 by increased level of Su(var)3-7 is dependent on Su(var)3-9 but appears only in some particular chromatin context.
Excess Su(var)3-7 recruits HP1 specifically to the male X chromosome
We know that Su(var)3-7 interacts with HP1 (Cléard et al., 1997; Delattre et al., 2000
), that Su(var)3-9 also interacts with HP1 (Aargard et al., 1999; Schotta et al., 2002
) and that Su(var)3-7 interacts with Su(var)3-9 according to two-hybrid data (Schotta et al., 2002
). The next question is how does Su(var)3-7 recruit the two others? We have determined that in the absence of Su(var)3-9 methyl-transferase, HP1 cannot be recruited on the X chromosome by overproduction of Su(var)3-7. This implies that H3-K9 methylation is a prerequisite for HP1 recruitment. Hence, we propose that Su(var)3-7 recruits first Su(var)3-9 on the male X chromosome and then HP1. Accumulation of H3-diMeK9 and HP1 on the male X chromosome is consistent with the finding that methylation of H3-K9 provides binding sites for HP1 (Bannister et al., 2001
; Lachner et al., 2001
; Jacobs et al., 2001
). It is interesting to note that new sites of H3-diMeK9 on autosomes and on the female X chromosomes do not efficiently recruit HP1 (within the limits of detection allowed by the technique). There might not be enough endogenous HP1 for recruitment at all new H3-diMeK9 sites. Alternatively, a certain amount of H3-diMeK9 may be necessary to efficiently recruit HP1, or the presence of H3-diMeK9 sites is not sufficient to recruit and maintain HP1 on chromosomes. A special chromatin environment existing on the male X chromosome seems critical for efficient HP1 targeting. Interestingly, Greil et al. (Greil et al., 2003
) have shown that HP1 and Su(var)3-9 can form different complexes and bind independently of each other at distinct sets of genes. Hence, access of HP1 to H3-diMeK9 may be blocked at some loci, owing to the chromatin environment or competition with other H3-MeK9-binding proteins. Our work reveals the existence of two types of heterochromatic complexes established on euchromatin: a complex made of Su(var)3-7, HP1 and H3-diMeK9 on the male X chromosome and a complex made of only Su(var)3-7 and H3-diMeK9 at some euchromatic loci on autosomes.
In summary, we have shown that the level of expression of Su(var)3-7 is critical for fly viability and integrity of chromosome morphology, that increased expression of Su(var)3-7 alone is sufficient to trigger heterochromatin formation by recruitment of its heterochromatic partners and that the male X chromosome is particularly sensitive to the dose of the protein. It will be interesting to determine which chromatin environment is prerequisite for efficient recruitment of Su(var)3-9 and HP1 by Su(var)3-7. The male X chromosome is of special interest given its particular chromatin environment and we are currently examining a potential link between the dosage compensation complex and Su(var)3-7.
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Acknowledgments |
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References |
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Aagaard, L., Laible, G., Selenko, P., Schmid, M., Dorn, R., Schotta, G., Kuhfittig, S., Wolf, A., Lebersorger, A., Singh, P. B. et al. (1999). Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18, 1923-1938.
Akhtar, A. (2003). Dosage compensation: an intertwined world of RNA and chromatin remodelling. Curr. Opin. Genet. Dev. 13, 161-169.[CrossRef][Medline]
Ayyanathan, K., Lechner, M. S., Bell, P., Maul, G. G., Schultz, D. C., Yamada, Y., Tanaka, K., Torigoe, K. and Rauscher, F. J., III (2003). Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. Genes Dev. 17, 1855-1869.
Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A., Thomas, J. O., Allshire, R. C. and Kouzarides, T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120-124.[CrossRef][Medline]
Cléard, F. and Spierer, P. (2001). Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein. EMBO Rep. 2, 1095-1100.
Cléard, F., Matsarskaia, M. and Spierer, P. (1995). The modifier of position-effect variegation Suvar(3)7 of Drosophila: there are two alternative transcripts and seven scattered zinc fingers, each preceded by a tryptophan box. Nucleic Acids Res. 23, 796-802.[Abstract]
Cléard, F., Delattre, M. and Spierer, P. (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.
Czermin, B., Schotta, G., Hulsmann, B. B., Brehm, A., Becker, P. B., Reuter, G. and Imhof, A. (2001). Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep. 2, 915-919.
Delattre, M., Spierer, A., Tonka, C. H. and Spierer, P. (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.
Eissenberg, J. C. and Elgin, S. C. (2000). The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev. 10, 204-210.[CrossRef][Medline]
Eissenberg, J. C., James, T. C., Foster-Hartnett, D. M., Hartnett, T., Ngan, V. and Elgin, S. C. (1990). Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 87, 9923-9927.
Eskeland, R., Czermin, B., Boeke, J., Bonaldi, T., Regula, J. T. and Imhof, A. (2004). The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43, 3740-3749.[CrossRef][Medline]
Greil, F., van der Kraan, I., Delrow, J., Smothers, J. F., de Wit, E., Bussemaker, H. J., van Driel, R., Henikoff, S. and van Steensel, B. (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.
Henikoff, S. (2000). Heterochromatin function in complex genomes. Biochim. Biophys. Acta 1470, 1-8.
Jacobs, S. A., Taverna, S. D., Zhang, Y., Briggs, S. D., Li, J., Eissenberg, J. C., Allis, C. D. and Khorasanizadeh, S. (2001). Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J. 20, 5232-5241.
James, T. C., Eissenberg, J. C., Craig, C., Dietrich, V., Hobson, A. and Elgin, S. C. (1989). Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J. Cell Biol. 50, 170-180.[Medline]
Jaquet, Y., Delattre, M., Spierer, A. and Spierer, P. (2002). Functional dissection of the Drosophila modifier of variegation Su(var)3-7. Development 129, 3975-3982.
Lachner, M., O'Carroll, D., Rea, S., Mechtler, K. and Jenuwein, T. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120.[CrossRef][Medline]
Li, Y., Kirschmann, D. A. and Wallrath, L. L. (2002). Does heterochromatin protein 1 always follow code? Proc. Natl. Acad. Sci. USA 99, 16462-16469.
Li, Y., Danzer, J. R., Alvarez, P., Belmont, A. S. and Wallrath, L. L. (2003). Effects of tethering HP1 to euchromatic regions of the Drosophila genome. Development 130, 1817-1824.
Nielsen, S. J., Schneider, R., Bauer, U. M., Bannister, A. J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R. E. et al. (2001). Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-565.[CrossRef][Medline]
Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M. and Nakatani, Y. (2002). A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296, 1132-1136.
Platero, J. S., Hartnett, T. and Eissenberg, J. C. (1995). Functional analysis of the chromo domain of HP1. EMBO J. 14, 3977-3986.[Abstract]
Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D. et al. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-599.[CrossRef][Medline]
Reuter, G. and Spierer, P. (1992). Position effect variegation and chromatin proteins. Bioessays 14, 605-612.[Medline]
Reuter, G., Giarre, M., Farah, J., Gausz, J., Spierer, A. and Spierer, P. (1990). Dependence of position-effect variegation in Drosophila on dose of a gene encoding an unusual zinc-finger protein. Nature 344, 219-223.[CrossRef][Medline]
Richards, E. J. and Elgin, S. C. (2002). Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108, 489-500.[Medline]
Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Jenuwein, T., Dorn, R. and Reuter, G. (2002). Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21, 1121-1131.
Schotta, G., Ebert, A., Dorn, R. and Reuter, G. (2003). Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila. Semin. Cell Dev. Biol. 14, 67-75.[CrossRef][Medline]
Schultz, D. C., Ayyanathan, K., Negorev, D., Maul, G. G. and Rauscher, F. J., III (2002). SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 16, 919-932.
Seum, C., Pauli, D., Delattre, M., Jaquet, Y., Spierer, A. and Spierer, P. (2002). Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster. Genetics 161, 1125-1136.
Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H. et al. (2002). G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779-1791.
Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G. and Reuter, G. (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.[Abstract]
Turner, B. M., Birley, A. J. and Lavender, J. (1992). Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375-384.[Medline]
Wallrath, L. L. (1998). Unfolding the mysteries of heterochromatin. Curr. Opin. Genet. Dev. 8, 147-153.[CrossRef][Medline]
Weiler, K. S. and Wakimoto, B. T. (1995). Heterochromatin and gene expression in Drosophila. Annu. Rev. Genet. 29, 577-605.[CrossRef][Medline]