HP1/Su(var)205

DEVELOPMENTAL BIOLOGY

Embryonic

There is an enrichment of HP1 in the intensely staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized to punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14. Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of HP1 is observed during mitosis in both early and late Drosophila embryos. Drosophila tissue culture cells prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes also show an enrichment for HP1 in the heterochromatin of the chromosomes (Kellum, 1995a).

HP1 is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent (James, 1989).

Cytological evidence is provided for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation (Fanti, 1998a).

Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, it has been shown that conditional depletion of HP1 in transgenic flies results in preferential lethality in male flies. Cytological analysis of mitotic chromosomes shows that HP1 depletion causes sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, are preferentially increased in males as well. Expression analysis shows that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes show a greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner (Liu, 2005).

Mutations in D. melanogaster Su(var)205 (also called HP1) cause lethality at larval stages, precluding a systematic functional analysis of Su(var)205 during development. To circumvent this limitation, the role of HP1 was studied using a Gal4-inducible RNA interference (RNAi) system, which allows for depletion of HP1 in a tissue- and developmental stage-specific manner. D. melanogaster y w^67c23 embryos were transformed with a construct expressing double-stranded RNA from a Su(var)205 cDNA. To deplete HP1, four independent transgenic lines (HP1-2, HP1-11, HP1-21 and HP1-31) were crossed with an act-Gal4 line (y w; +/+; act-Gal4/TM6B) expressing Gal4 ubiquitously during development. Resulting larval progeny from lines HP1-11 and HP1-21 showed a reduction in HP1 levels of ~90%, line HP1-31 showed a 60% reduction and line HP1-2 showed no reduction. Those progeny with a 60%-90% reduction in HP1 generally survived to the third-instar larval stage, but progeny with a 90% reduction rarely survived to the adult stage. Lethality mainly occurred at the pupal stage and seemed to be due to a failure to eclose. Adult progeny of the HP1-31/act-Gal4 line were also viable, but the female:male ratio was highly skewed (2.4:1 versus 0.9:1 for this genotype at the larval stage). An alteration in the sex ratio was also evident in adult flies from the HP1-11/act-Gal4 line: all 21 survivors were female. There were no adult HP1-21/act-Gal4 survivors when progeny were grown at 25°C, but 30 'escapers' were obtained at 18°C, all of which were female. Collectively, these results suggested that there was an association between sex-biased lethality and HP1 dosage (Liu, 2005).

To assess the cause of lethality on depletion of HP1, lines HP1-21 and HP1-11 were crossed with lines inducing RNAi exclusively in eye imaginal discs (ey-Gal4) and in the posterior compartment of developing wings (en-Gal4) and the effect of HP1 depletion in these tissues was examined in third-instar larvae. In HP1-depleted imaginal discs, an increased number of dying cells was consistently found using acridine orange staining, which is often used to detect apoptotic cells. Tissue growth defects were also observed in the eyes and wings of adult flies; defects in both of these tissues were more severe in males than in females. Apoptosis seemed to be mediated through a caspase-dependent pathway; tissue growth defects could be partially rescued by the addition of p35, a cysteine protease apoptosis inhibitor. These results suggest that the observed lethality and growth defects in both sexes are linked to apoptosis (Liu, 2005).

It was next asked whether sex-specific lethality involves specific mitotic chromosome defects, as observed previously in Su(var)205 mutants. A variable number of 'ring-like' chromosomes and other aberrant segregated chromosomes (e.g., chromatin bridges) was found in the metaphase spreads from third-instar larval neuroblast cells of HP1-depleted larvae. The relative frequency of defective mitotic chromosomes in HP1-depleted males was approximately twice that in females, indicating that differential chromosomal segregation defects may underlie sex-biased lethality (Liu, 2005).

Because the mutated lethal allele Su(var)205⁰² does not result in telomeric fusions, however, lethality cannot be solely due to this cause. To explore whether additional mechanisms are involved in the sex-biased lethality, the impact of HP1 depletion on core histone modifications was measured, since increases in histone acetylation can cause apoptosis. Using cell extracts from larval imaginal discs of HP1 RNAi mutants and control larvae, the global levels were compared of several core histone modifications in males and females. The levels of acetylation at Lys8 of histone H4 (H4K8ac), methylation at Lys4 of histone H3 (H3K4me) and methylation at Lys79 of histone H3 (H3K79me; all hallmarks of active chromatin) were all increased in males after HP1 depletion. But levels of methylation at Lys20 of histone H4 (H4K20me) and methylation at Lys9 of histone H3 (H3K9me; both hallmarks of heterochromatin) showed a global decrease when cells were lysed in 300 mM salt buffer. No change was observed in H4K20me or H3K9me when cells were lysed in SDS buffer, suggesting that the changes in histone modifications associated with the active state may have a role in the observed lethality. These effects were not caused by misregulation of genes encoding known histone-modifying enzymes, including histone methylases, acetylases or deacetylases, as these were unaffected by HP1 depletion (Liu, 2005).

It was asked whether any change in histone H3K9me occurs on chromatin, since this histone modification is interdependent with the dynamics of HP1. In polytene chromosomes from HP1-depleted mutants, H3K9me remains at the pericentric heterochromatin region in both sexes. But the intensity of the pericentric H3K9me signal in males is lower than that in females, a modification linked to X-chromosome dosage compensation in males. This staining showed no obvious changes that were dependent on HP1. To test the possibility that the HP1-induced preferential lethality in males is linked to the disruption of specific functional genes in males, total RNA isolated from two independent populations of male and female third-instar larvae of line HP1-21/act-Gal4 was compared using microarray analysis. More than 200 predicted transcripts or genes were specifically affected in males, but only 119 were specifically affected in females; 127 genes seemed to be affected in both males and females. Among the affected genes with known function, those essential for DNA replication, such as mus209 and Mcm6, were downregulated in both sexes; wrinkled (W) and Rep4, both regulators of apoptosis, were upregulated. Notably, a number of genes encoding cell cycle regulators, such as fizzy (fzy), pimples (pim), cyclin-dependent kinase subunit (Cks30A) and the DNA replication initiation inhibitor geminin, were all specifically affected only in males, suggesting that these genes have a role in the observed differential lethality. Transcription of genes known to regulate the sex ratio, such as msl-1 (also called MSL), was not affected (Liu, 2005).

Of the 127 genes affected in both males and females, transcription of nearly two-thirds were upregulated in the absence of HP1. In addition, 22 of 24 genes that had lower expression in wild-type females were upregulated in the female RNAi mutants. These observations are consistent with the known role of HP1 in transcriptional gene silencing. In addition, however, nearly one-half of the affected genes were downregulated in the absence of HP1, supporting the possibility that HP1 may have a role in both negative and positive regulation of transcription (Liu, 2005).

To determine whether the sex-biased effects of HP1 on histone modification and transcription were due to a differential distribution of HP1 on chromatin in males and females, chromatin immunoprecipitation (ChIP) analysis was carried out. Sonicated chromatin extracts of nuclei isolated from male and female third-instar y w^67c23 larvae were immmunoprecipitated with polyclonal antibodies against D. melanogaster HP1. Among the eight genes tested that were affected in both males and females, four showed similar levels of HP1 binding enrichment in the two sexes, implying a direct role for HP1 in their transcription. Of 12 genes specifically affected in females, six were enriched in HP1 binding to similar levels in both sexes, and the other six were HP1-negative. Notably, 11 of 18 genes specifically affected in males showed a severalfold enrichment of HP1 binding in males compared with females; 5 were similarly enriched in both sexes, and 2 were not associated with HP1. Although the ChIP results indicated that genes specifically affected in males seemed to be enriched in HP1 binding in males compared with females, genes specifically affected in females did not have a 'female-specific' HP1 binding pattern, indicating that HP1 might invoke sex-specific mechanisms in the regulation of chromatin or transcription (Liu, 2005).

These results show that HP1 has rather different roles in males versus females. RNAi knock-down of HP1 resulted in sex-biased defective chromosome segregation, alterations in histone modifications, specific changes in transcription and a skewed sex ratio in surviving progeny. Two recent studies suggest that chromosomal segregation defects, particularly telomeric fusion, may have a key role in the apoptosis and sex-biased lethality observed in this study. Overexpression of the heterochromatin protein Su(var)3-7 also induces lethality in males, with a shortened or condensed X chromosome. But the morphology of the X chromosome and the global level and distribution of H4K16ac seem unaffected in male HP1 knock-down progeny, suggestive of an alternative mechanism (Liu, 2005).

The differential change in H3K9me on chromatin may be due to an alteration in Su(var)3-9 localization, since HP1 is essential for maintaining its dynamics. Changes in global histone acetylation and phosphorylation could result from an HP1-induced global change in chromatin structure or from secondary effects; the absence of a Su(var)3-9 homolog in mammals also caused changes in different histone modifications, in addition to H3K9me. Notably, all these changes occur in a sex-biased manner. This is attributed to the sex-specific distribution of HP1 on chromatin, shown by ChIP analysis. This hypothesis suggests that the male genome, relatively enriched in HP1, is subject to more changes in histone modifications, more chromosome segregation defects and more changes in transcription in the absence of HP1, which seems to be the case. The heterochromatic Y chromosome in males may be also involved in the sex-biased distribution of HP1 in the genome, for example, by altering the distribution of remaining HP1 and other heterochromatin proteins (Liu, 2005).

A previous cytological study of mealybugs identified a conspicuous HP1-associated 'mass/aggregate' structure in male chromosomes, contrasting with a scattered localization along female chromosomes. This result and the results presented in this study support the hypothesis that HP1 has a distinct regulatory role in male versus female chromatin. Whether the sex-specific distribution of HP1 on chromatin directly regulates the sex-biased differences in global transcription, showing relatively lower transcription in males than in females, is not known. The facts that HP1 is involved in transcriptional gene silencing and that depletion of HP1 results in upregulation of some male genes, normally transcribed at a lower level in males than in females, seem to support the idea that HP1 has a role in the phenomenon. But these sex-biased regulation mechanisms also seem to require other sex-specific regulators (e.g., proteins or RNA). Future studies are required to define those regulators and to understand their role in the organization of sex-biased chromatin and transcriptional regulation. Understanding the mechanisms of this regulation may also yield important clues about the basis of sexual dimorphism in animals (Liu, 2005).

Effects of Mutation or Deletion

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. The subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c) have been characterized, comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. Truncated and chimeric HP1 proteins were expressed in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. It is concluded that sequence variations within the hinge and shadow account for HP1 targeting distinctions. It is proposed that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins (Smothers, 2001).

Two allelic dominant suppressors of position-effect variegation (PEV) are found to contain mutations within the gene encoding HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for nonsense codon, a truncated HP-1 protein is detected by western blot analysis. An HP-1 minigene under control of an Hsp70 heat-inducible promoter, was transduced into flies by germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression appear to depend upon the level of expression of this heterochromatin-specific protein. It is thought that PEV arises from alterations in mass action-drive heterochromatin assembly and a requirement for a precise stoichiometry of heterochromatin protein subunits (Eissenberg, 1992 and references).

Point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing (Platero, 1995).

Heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7-8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos (Eissenberg, 1993).

The insertion of a heterochromatin segment into a euchromatic gene (brown, an eye color locus), results in position-effect variegation of brown. The insertion of heterochromatin also causes the aberrant cytological association of the gene and its homologous copy to heterochromatin. The cytological association of the heterochromatic region is affected by chromosomal distance from heterochromatin and by genic modifiers of PEV. Thus HP1 mutations, which can result in position-effect variegation, suppress trans-inactivation of the heterochromatinized euchromatic gene. When HP-1 is present in three doses, PEV of brown is enhanced (Csink, 1996).

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allows for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV (Cryderman, 1999).

The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1. The Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. Heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes (Lu, 2000).

Both the dominant and recessive phenotypes of mutations in HP1 were examined to look for an essential requirement for HP1 in development. It is proposed that reduced expression of one or more essential heterochromatic genes results in the recessive late larval lethality of Su(var)2-5. In support of this hypothesis, the essential heterochromatic genes rolled and light are misregulated in Su(var)2-5 mutants. rolled transcription at its normal chromosomal location is reduced in Su(var)2-5 mutant flies. Since no maternal Rolled protein is detectable in third instar larvae homozygous for rolled deficiencies, the RNA levels that are detected in mutant larvae and adults reflect zygotic gene expression. In the case of the heteroallelic mutant larvae, it should be emphasized that at the time the larvae were collected for Northern analysis, the Su(var)2-5 mutant larvae appeared healthy and would have lived on for several more days as third instar larvae before dying; indeed, a further decline in rolled RNA preceding larval death cannot be ruled out. Thus, reduced expression of rolled could contribute to the defects associated with loss of HP1. Of course, reduced expression of other heterochromatic genes probably also contributes to lethality due to HP1 deficiency (Lu, 2000 and references therein).

light also experiences variegated inactivation in Su(var)2-5 larval Malpighian tubules, and light transcripts are dramatically reduced overall in Su(var)2-5 mutant larvae. It is important to stress that the repressed light locus in these experiments is also in its normal chromosomal location. It is concluded that silencing of light in these experiments is a direct consequence of HP1 depletion, depriving the light locus of the heterochromatin context required for its normal expression. Several other genes reside in heterochromatin, and it will be interesting to see whether dependence on HP1 is a general attribute of gene expression in heterochromatin (Lu, 2000).

Mutations in rolled, like Su(var)2-5 mutations, lead to late larval or early pupal lethality with defective or missing imaginal discs. At the cytological level, rolled mutations cause defects in mitosis, including overcondensed and/or lagging anaphase chromosomes. Intriguingly, neuroblasts of larvae doubly mutant for hypomorphic alleles of rl and abnormal spindles (encodes a microtubule-associated protein) show telomeric stickiness and increased frequency of aneuploid mitotic figures. These phenotypes are also seen in neuroblasts of larvae heteroallelic for Su(var)2-5 mutations; indeed, the highest frequency of defects occurs in larvae heteroallelic for the Su(var)2-5²⁰⁵ allele, which is carried on a chromosome marked with a hypomorphic rl allele. Therefore, reduced expression of rolled caused by loss of HP1 could contribute to mitotic defects in HP1 mutant larval brains (Lu, 2000).

How can HP1 be required both for activation of heterochromatic genes and silencing of euchromatic genes? It has been proposed that certain heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when heterochromatic genes are deprived of such essential heterochromatic proteins by displacement away from heterochromatin 'compartments' where such proteins are in high concentration. Such context-dependent regulatory activity has also been described for yeast RAP1 (repressor/activator protein 1); RAP1 is required for high-level expression of many ribosomal protein and glycolytic enzyme genes, but it promotes position-effect silencing at the HM silent mating type cassettes and telomeres. Genetic evidence suggests that RAP1 has distinct activator and silencing domains that could recruit or stabilize distinct chromosomal complexes at distinct chromosomal sites. Similarly, HP1 could interact with different proteins or protein complexes to promote silencing or activation in different chromosomal contexts. Another possibility is that HP1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted and dependent. Loss of HP1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context (Lu, 2000).

HP1 is required for correct chromosome segregation in Drosophila embryos (Kellem, 1995b). HP1 has been reported to cause recessive embryonic lethality associated with defects in chromosomal morphology and mitotic segregation (Kellem, 1995b). The conclusion reached by Kellum (1995b) that HP1/Su(var)2-5 is a recessive embryonic lethal has been reported to be incorrect. An intimation of this may be found in the Fanti (1998) where they mention in passing that they recovered about 20% heteroallelic Su(var)2-5 mutant third instars (indeed, this whole paper would have been impossible if Su(var)2-5 were a recessive embryonic lethal, since the cytology involves third instar neuroblasts). For five different Su(var)2-5 alleles in six different allelic combinations, heteroallelic flies survive in Mendelian proportions to the end of third instar, and die at or prior to pupariation. This was done using genetically marked larvae. Kellum and Alberts (1995b) finding cannot at this time be explained, but their conclusion was based on the observation of a reduced hatch rate and the assumption that the unhatched embryos must be HP1 homozygous mutant. In unpublished work, Eissenberg reports also finding mitotic defects in flies from such crosses, but using blue balancers, could show that the mitotic defects were not correlated with genotype (Lu, 2000).

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. A genetic system has been developed which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. The Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in Drosophila (Savitsky, 2002).

Two highly conserved histone deacetylases, Sir2 and Rpd3, have been linked to caloric restriction and the extension of longevity. Because the Drosophila forms of each protein can silence genes in either euchromatin or heterochromatin, it was determined whether longevity extension is mediated by silencing in the latter domain. When silencing was increased and decreased using mutations that affect heterochromatin protein 1 (HP1), but have no direct effect upon Sir2 or Rpd3, lifespan was unaffected. Heterochromatin-mediated gene silencing was then modulated without directly influencing HP1 as well as the deacetylases, again yielding no effect on lifespan. Mortality rates were unchanged by all manipulations, indicating that euchromatic targets are likely to be the effectors of deacetylase-mediated longevity extension in Drosophila (Frankel, 2005).

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, a specific bloating of the male X chromosome is observed, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose (Spierer, 2005).

Su(var)3-7 function is still poorly understood. It encodes a large protein associated with pericentric heterochromatin, telomeres and a few euchromatic sites on interphase polytene chromosomes. Seven widely spaced zinc fingers stand out in the sequence of the N-terminal half. In vitro, the zinc finger region of Su(var)3-7 has affinity for DNA, and preferentially for some satellite sequences. There is also evidence for direct binding of Su(var)3-7 with DNA in vivo. The N-terminal half of Su(var)3-7 interacts nonspecifically in vivo with heterochromatin and euchromatin, whereas the C-terminal half promotes interaction with itself, and with pericentric heterochromatin. Su(var)3-7 also interacts genetically and physically with HP1 and with Su(var)3-9, as determined in yeast by the two-hybrid assay and in vivo. To decipher the function of Su(var)3-7, mutants were generated by homologous recombination, and a detailed examination was undertaken of their phenotype. Su(var)3-7 was shown to be essential, the maternal contribution being sufficient for viability. Interestingly, males are more sensitive than females to the lack of Su(var)3-7. The cause of this lethality is unknown (Spierer, 2005).

This study reports the building of a new mutant of Su(var)3-7 by homologous recombination: described are the phenotypes of mutations on polytene chromosome morphology and on the organism -- these are similar to phenotypes resulting from mutational loss of HP1. The male X chromosome is more sensitive to these effects, leading to an understanding of an interaction between the modifier of PEV Su(var)3-7 and the dosage compensation machinery. It is concluded that the importance of the roles and partnership of Su(var)3-7 and HP1 extend beyond genomic silencing in the maintenance of chromosome integrity and function, including the male X-specific chromosome-wide mechanism of dosage compensation (Spierer, 2005).

Polytene chromosomes are affected similarly by severe loss of Su(var)3-7 or HP1. In both cases, the main mutant phenotype is a bloated X in males, and an expanded chromocenter in males and females. Why is chromosome morphology modified when HP1 or Su(var)3-7 amounts are strongly reduced? There are several possible explanations. Su(var)3-7 and HP1 are both required for stability of chromatid association, and reduction of dose could lead to dissociation. This mechanism has been suggested for similar phenotypes in other conditions. This hypothesis could be tested by determining whether a phenomenon based on chromatid association, such as transvection, is affected in Su(var)3-7 or HP1 mutants. Su(var)3-7 and HP1 are required for compaction of intercalary heterochromatin on euchromatic arms. The loss of this compaction, similar to what is seen at the chromocenter, could lead to bloating and disruption of the banding pattern. If indeed Su(var)3-7 and HP1 are instrumental in chromosome compaction, then one could expect that excess amounts of the proteins lead in turn to an excess of compaction. This is actually the case for Su(var)3-7; increasing amounts of Su(var)3-7 first affect the male X chromosome, which becomes strongly compacted. Furthermore, targeting HP1 to an ectopic site promotes chromosomal loops linking this ectopic site with sites of intercalary heterochromatin. The question remains of the particular sensitivity of the male X chromosome to loss and excess of Su(var)3-7 and to loss of HP1 (Spierer, 2005).

That the male X chromosome is affected first and most severely could result from association of this chromosome with the dosage compensation complex (DCC). Chromatin relaxation triggered by the DCC in the male X would render it more sensitive to variations of the amount of chromatin-associated proteins. Indeed, male X bloating and shortening has been observed in several conditions, and has been named the 'pompon' phenotype and described as resulting from specific environmental aggressions or mutations. Male X bloating was described as resulting from the loss of several chromatin-modifying factors such as Jil-1 or the Nurf complex. The various environmental and genetic conditions in which bloating of the male X occurs underline the peculiar sensitivity of the phenotype, and could explain the differences of phenotype intensity seen using different X chromosomes (Spierer, 2005).

Finally, the X-chromosome-specific phenotype might result from a direct interaction between the DCC and silencing factors. This paper indeed demonstrates a genetic interaction between an essential gene of the dosage compensation machinery, mle, and Su(var)3-7. However, in the wild type, preferential association of Su(var)3-7 with the polytene male X chromosome has not been detected using either a polyclonal antibody raised against Su(var)3-7 sequences, or a monoclonal antibody raised against the tag of HA-Su(var)3-7. However, preferential association with the male X is seen when Su(var)3-7 is over-expressed from a transgene. At this point it is not possible to distinguish between two possibilities: either Su(var)3-7 modulates the transcription level of the X chromosome by counteracting the DCC relaxing effect, or it protects the X-linked genes that do not need to be dosage compensated. The role of HP1 also remains to be explored. No preferential association of HP1 with the male X polytene chromosome has been seen. Nevertheless, when Su(var)3-7 is over-expressed, HP1 is found associated preferentially with the male X (Spierer, 2005).

In conclusion, Su(var)3-7 and HP1 participate in chromocenter and male X polytene chromosome integrity. The similarity of the phenotypes seen in mutations of either one, the partial compensation of the loss of dose in one by an increase of dose in the other in PEV, and the physical interaction between Su(var)3-7 and HP1 seen in vitro and in vivo (Delattre, 2000) all point to the same conclusion. These two structurally very different proteins cooperate closely in chromosome organization. An interaction also existes between Su(var)3-7 and compensation of dose. This interaction between the genomic silencing of PEV dependent on Su(var)3-7 association, and hyperactivation dependent on association of the DCC, needs to be unravelled (Spierer, 2005).

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