In order to examine in living tissue the dynamics of JIL-1 distribution throughout the cell cycle, transgenic fly lines expressing a green fluorescent protein, JIL-1 fusion protein (JIL-1-GFP), were generated. JIL-1 containing GFP coding sequence at its NH2 terminus was constructed in the pCaSpeR hs83 P element vector and used to generate 14 independent lines of transgenic flies. Although all lines show the same localization pattern, the strongest fluorescing line was selected for further study and crossed into a background containing the vin5 deficiency, which removes one copy of the JIL-1 gene, increasing the proportion of detectable JIL-1-GFP product. Immunoblotting of transgenic embryonic proteins with JIL-1 antibody reveals an additional protein band not present in wild-type flies that is consistent with the predicted size of the JIL-1-GFP protein. This indicates that the fusion protein could readily be detected on immunoblots and that it was being stably expressed (Jin, 1999).
To study the cellular localization of the JIL-1-GFP fusion protein, various tissues, including ovaries, imaginal discs, third instar larval bodywall muscles, and salivary glands, were dissected and imaged live in physiological saline using confocal microscopy. In all tissue examined, JIL-1-GFP is detectable at high levels in the nucleus approximately 4 hr after heat shock induction. The JIL-1-GFP fusion protein is clearly localized to the nucleus of all the cells, including the nuclei of the large polyploid nurse cells and the smaller diploid follicle cells. The JIL-1-GFP fluorescence in the nucleus is not uniform but is organized in a discrete pattern that overlapped with Hoechst fluorescence in double-labeled preparations, suggesting that JIL-1 is localized to chromosomes in the nucleus (Jin, 1999).
JIL-1-GFP distribution was imaged during the cell cycle in early syncytial blastoderm embryos. Time lapse movies were generated from confocal sections obtained with 20 s intervals from live embryos. JIL-1-GFP is widely distributed throughout the nucleus at interphase, condenses during prophase, aligns at the metaphase plate, and moves to the spindle poles at anaphase, where its distribution then decondenses back into the interphase pattern. This sequence of events is very similar to chromosomal dynamics during the cell cycle as observed in Drosophila by video time lapse of fluorescently tagged histone and suggests that the JIL-1 nuclear kinase is chromosomally localized throughout the cell cycle. No indications of dominant-negative effects were observed as a consequence of the overexpression of the JIL-1-GFP construct (Jin, 1999).
JIL-1's distribution pattern was analyzed in confocal images of larval polytene chromosomes. Chromosomal squashes prepared from salivary glands of climbing third instar larvae were stained with JIL-1 antisera and double-labeled with Hoechst to visualize the DNA. Hoechst staining is brightest in the condensed banded regions of the chromosome due to the higher concentration of DNA. Conversely, Hoechst signal is weaker or absent in the gene-rich interband regions that are comprised of less tightly packed euchromatin. The results obtained in a chromosomal squash from a female larva demonstrate that JIL-1 localizes to hundreds of sites along the polytene chromosome thatcorrespond to interband regions, and that this staining shows only a very limited overlap with regions of strong Hoechst labeling. Since interbands arise from partial unfolding of the 30 nm chromatin fiber and have been proposed to be the sites of actively transcribed genes, these findings suggest that JIL-1 may be potentially involved in gene activity by regulation of chromatin structure via histone phosphorylation. However, it is clear that JIL-1 is not required at all locations of decondensed chromatin since there are interband regions that do not show JIL-1 labeling (Jin, 1999).
To test the hypothesis that JIL-1 expression and localization may be correlated with transcriptional regulation, the expression of JIL-1 on female and male X chromosomes, respectively, was tested in relation to JIL-1 expression on autosomes. The unpaired male X contains only half the amount of DNA as the paired female X chromosomes and the autosomes, and to compensate for this, the transcriptional level of the male X is upregulated about 2-fold. X chromosomes are much more intensely labeled than autosomes, although the banding pattern is maintained. In contrast Hoechst binding to the X chromosome is much less than to the autosomes. To verify the localization pattern of JIL-1 obtained by antibody labeling, live polytene nuclei from JIL-1-GFP transgenic flies were imaged using confocal microscopy. JIL-1-GFP is localized on all chromosomes in a banded pattern similar to that observed by antibody labeling. Furthermore, the level of JIL-1-GFP in live transgenic animals is also upregulated on the male X as compared to autosomes (Jin, 1999).
In order to quantitate the difference in labeling of the X chromosomes in males and females, the average pixel intensity of JIL-1 immunostaining for X chromosomes was determined in males and females and it was compared to the autosomal staining intensity, which was normalized to a value of 1.0. In female polytene squashes there is no significant difference between autosomal and X chromosome staining intensity, whereas in males, a highly statistically significant difference is found between autosomal and X-chromosomal staining intensities. There is an almost 2-fold increased level of JIL-1 on the Drosophila male X chromosome compared to autosomes, which correlates well with the roughly 2-fold increased transcription level on this chromosome due to dosage compensation mechanisms. These results support a model whereby JIL-1 activity is involved in regulating gene expression (Jin, 1999).
Histone lysine methylation is an epigenetic mark to index chromosomal subdomains. In Drosophila, H3-K9 di- and trimethylation is mainly controlled by the heterochromatic SU(VAR)3-9 HMTase, a major regulator of position-effect variegation (PEV). In contrast, H3-K27 methylation states are independently mediated by the Pc-group enzyme E(Z). Isolation of 19 point mutants demonstrates that the silencing potential of Su(var)3-9 increases with its associated HMTase activity. A hyperactive Su(var)3-9 mutant, pitkinD, displays extensive H3-K9 di- and trimethylation within but also outside pericentric heterochromatin. Notably, mutations in a novel Su(var) gene, Su(var)3-1, severely restrict Su(var)3-9-mediated gene silencing. Su(var)3-1 was identified as an 'antimorphic' mutant of the euchromatic H3-S10 kinase JIL-1. JIL-1Su(var)3-1 mutants maintain kinase activity and do not detectably impair repressive histone lysine methylation marks. However, analyses with seven different PEV rearrangements demonstrate a general role of JIL-1Su(var)3-1 in controlling heterochromatin compaction and expansion. These data provide evidence for a dynamic balance between heterochromatin and euchromatin, and define two distinct mechanisms for Su(var) gene function. Whereas the majority of Su(var)s encode inherent components of heterochromatin that can establish repressive chromatin structures [intrinsic Su(var)s], Su(var)3-1 reflects gain-of-function mutants of a euchromatic component that antagonize the expansion of heterochromatic subdomains [acquired Su(var)s] (Ebert, 2004).
The identification of Su(var) genes in Drosophila reveals many important proteins regulating higher-order chromatin structure. SU(VAR)3-9, HP1, and SU(VAR)3-7 are inherent components of heterochromatin that can establish and maintain a heterochromatic chromatin structure. By genetic means, Su(var)3-9 is the dominant component over Su(var)2-5 (HP1) and Su(var)3-7, indicating a major function of the associated HMTase in the formation of repressive chromatin regions (Ebert, 2004 and reference therein).
In Drosophila, heterochromatic gene silencing is mainly determined by H3-K9 dimethylation. In agreement with its in vitro HMTase activity, Su(var)3-9 regulates H3-K9 di- and trimethylation at pericentric heterochromatin. In addition, other H3-K9-specific HMTases must exist that mediate H3-K9 dimethylation at the fourth chromosome and in the chromocenter core of Su(var)3-9 mutants. HP1 is another important control factor for H3-K9 methylation because in Su(var)2-5 mutants, H3-K9 mono- and dimethylation are dramatically increased along chromosomal arms. Although SU(VAR)3-9 shows extensive association with heterochromatic and euchromatic regions in HP1-null cells, the shift in these methylation patterns could also be mediated by other H3-K9-specific HMTases. At pericentric heterochromatin, H3-K9 methylation states appear unaltered; however, a slight reduction of H3-K9 dimethylation that is not detectable by immunofluorescence might be the cause of the HP1 Su(var) effect. Alternatively, impairment of H4-K20 trimethylation at pericentric heterochromatin could cause the loss of silencing in HP1 mutants (Ebert, 2004).
The analysis of Su(var)3-9 mutants has revealed novel hypomorphic and null alleles that show differential effects on H3-K9 methylation states. A direct correlation between their HMTase activities and the amount of gene silencing demonstrates that establishment of heterochromatic structures is to a large extent determined by the kinetic properties of the SU(VAR)3-9 HMTase reaction. Consistently, hyperactive SU(VAR)3-9ptn induces enhanced H3-K9 di- and tri-methylation, concomitant with an expansion of heterochromatin. This results in severe phenotypes such as dominant female sterility caused by overcompaction of the whole chromatin in early embryos. In this study it is shown that Su(var)3-9ptn mutants display around 100-200 additional bands with enhanced H3-K9 di- and tri-methylation in the arms of polytene chromosomes. These bands likely correspond to endogenous SU(VAR)3-9-binding sites and demonstrate that hyperactive SU(VAR)3-9ptn can mediate the expansion of repressive structures also at chromosomal arms (Ebert, 2004).
A loss-of-function Su(var) mutant reduces gene silencing by removing one or several intrinsic components of heterochromatin. Su(var)3-9ptn induces enhanced gene silencing, and it was therefore surprising to encode a mutant of a 'classical' Su(var) gene. Based on functional characterization, this mutant was identified as the first hypermorphic (gain-of-function) allele of an intrinsic Su(var) gene. Many more PEV modifier mutants that cause enhanced gene silencing have been isolated. Molecular characterization of some of these genes have revealed components of active chromatin, such as transcription factors; however, further characterization of this class of genes might also uncover other hypermorphic mutants of intrinsic Su(var) genes and contribute to the understanding of heterochromatin regulation (Ebert, 2004).
The mutant screen to identify genes that impair Su(var)3-9-mediated gene silencing led to the molecular identification of Su(var)3-1, the strongest Su(var) gene to date. Su(var)3-1 can restrict heterochromatin formation and generally dominate over the strong silencing effect of Su(var)3-9 extra copies. Su(var)3-1 codes for C-terminal truncations of the H3-S10 kinase JIL-1 that does not affect the JIL-1 ability to phosphorylate H3-S10 but selectively impairs the expansion of heterochromatin (Ebert, 2004).
Intrinsic Su(var) genes impair or weaken the heterochromatic structure. Loss-of-function Su(var)3-9 alleles or mutations in HDAC1 reduce H3-K9 dimethylation, whereas HP1 mutants impair H4-K20 trimethylation at pericentric heterochromatin. JIL-1 is not an intrinsic component of heterochromatin and does not affect any of the known repressive histone lysine methylation marks at pericentric heterochromatin. Nevertheless, JIL-1Su(var)3-1 alleles display a strong Su(var) effect. Therefore JIL-1Su(var)3-1 is categorized as a novel type of mutant with acquired Su(var) function. In contrast to intrinsic Su(var)s that encode components directly involved in heterochromatin formation, acquired Su(var) mutants would antagonize heterochromatin formation by stabilizing the euchromatic state or preventing heterochromatin expansion (Ebert, 2004).
The data assign a novel function to the C terminus of the JIL-1 kinase that is independent of its H3-S10 kinase activity. Lack of the C terminus could prevent phosphorylation or interaction with an as-yet-unknown target protein. Alternatively, the C terminus could comprise control functions that prevent excessive phosphorylation of this target. There are many possible target proteins that could be involved in higher-order chromatin formation. For example, the striking X-chromosome decondensation phenotype of JIL-1Su(var)3-1 mutants has also been described for iswi and nurf301, subunits of the NURF complex. Particularly, nurf301 mutants display a dominant Su(var) effect, indicating that chromatin remodeling processes are involved in the regulation of heterochromatin. Another possible target of the JIL-1Su(var)3-1 kinase might be the histone variant H2A.Z. In budding yeast, H2A.Z is required to prevent spreading of heterochromatin into euchromatin. This protective function might be modulated by JIL-1Su(var)3-1-mediated phosphorylation of H2A.Z. Finally, some intrinsic Su(var)s such as SU(VAR)3-9 and HP1 are phosphoproteins, and JIL-1Su(var)3-1-mediated phosphorylation of those proteins might affect their function in heterochromatin expansion (Ebert, 2004).
Based on these data, a model is proposed for a dynamic balance between euchromatin and heterochromatin. In PEV rearrangements, the boundary between these two states is determined by the antagonistic functions of euchromatic regulators (e.g., JIL-1) and the SU(VAR)3-9 HMTase that mediates H3-K9 dimethylation, the major mark of heterochromatin. The boundary between euchromatin and heterochromatin is not static and depends on the activity and abundance of inherent components of heterochromatin, such as, for example, hyperactive SU(VAR)3-9ptn or overexpression of Su(var)3-9. In contrast, hypoactive or null mutants of Su(var)3-9 weaken heterochromatin formation and favor the propagation of the euchromatic state. In JIL-1Su(var)3-1 mutants, heterochromatin expansion is severely repressed, even under conditions of elevated Su(var)3-9 function, and is most likely antagonized by currently unknown proteins that are potential targets of the gain-of-function JIL-1Su(var)3-1 kinase. Dependent on the phosphorylation state of these targets, either heterochromatin expansion can be blocked or the propagation of the euchromatic state is highly stabilized (Ebert, 2004).
This work significantly extends mechanistic insights into the formation of heterochromatin, and Su(var)3-1 was discovered as a novel class of PEV modifiers that controls the balance between euchromatic and heterochromatic subdomains. Because of the dominant role of JIL-1Su(var)3-1 over major components of heterochromatin [intrinsic Su(var)s], the putative JIL-1 mutant targets are predicted to have important functions in gene silencing and the higher-order structuring of chromatin. The ongoing analysis on the full definition of Su(var) gene function is therefore likely to reveal both the genetic and molecular hierarchy that dictates epigenetic gene control (Ebert, 2004).
A cytological and genetic characterization is provided of the JIL-1 locus in Drosophila. JIL-1 is an essential chromosomal tandem kinase and in JIL-1 null animals chromatin structure is severely perturbed. A range of JIL-1 hypomorphic mutations form an allelic series. JIL-1 has a strong maternal effect and JIL-1 activity is required at all stages of development, including embryonic, larval, and pupal stages. Furthermore, a new allele of JIL-1, JIL-1h9 is described that encodes a truncated protein missing COOH-terminal sequences. Remarkably, the truncated JIL-1 protein can partially restore viability without rescuing the defects in polytene chromosome organization. This suggests that sequences within this region of JIL-1 play an important role in establishing and/or maintaining normal chromatin structure. By analyzing the effects of JIL-1 mutations evidence is provided that JIL-1 function is necessary for the normal progression of several developmental processes at different developmental stages such as oogenesis and segment specification. It is proposed that JIL-1 may exert such effects by a general regulation of chromatin structure affecting gene expression (Zhang, 2003: full text of article).
The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. A detailed ultrastructural and cytological analysis of has been performed of the defects in JIL-1 mutant chromosomes. All autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes a model is proposed in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions (Deng, 2005).
A reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. Evidence is provided that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, a model is proposed where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization (Zhang, 2006: full text of article).
According to the histone code hypothesis and the recently proposed binary switch model, phosphorylation of a site adjacent to a methyl mark that engages an effector molecule may regulate its binding. JIL-1 phosphorylates the histone H3S10 residue in euchromatic regions of polytene chromosomes, raising the possibility that this phosphorylation at interphase prevents the recruitment of Su(var)3-9 and/or the dimethylation of the neighboring K9 residue. This, in turn, would affect the binding of HP1, thus antagonizing the formation of silenced heterochromatin at interbands. That different regions of chromatin may have different combinations of posttranslational modifications controlling effector/histone interactions, as predicted by the histone code hypothesis, is underscored by the finding that, in JIL-1 null backgrounds, the level of the dmH3K9 marker and HP1 are preferentially increased on the male and female X chromosomes. It is well documented that the male X chromosome is unique because of the activity of the MSL dosage compensation complex and the MOF histone acetyltransferase, which leads to hyperacetylation of histone H4. However, comparable markers for the female X chromosome have yet to be discovered, and the results are the first indication that markers may exist that distinguish male and female X chromosomes from autosomes, and that this difference may increase the affinity for Su(var)3-9. That the spreading of heterochromatic markers in the absence of JIL-1 occurs on both the male and female X chromosome further indicates that these changes are independent of dosage compensation processes (Zhang, 2006).
Unfortunately, in this study, the possibility that the observed spreading of heterochromatin markers occurred preferentially to specific euchromatic sites could not directly addressed. In JIL-1 null and hypomorphic backgrounds, chromosome morphology is greatly perturbed, and there is an intermixing not only of euchromatin and the compacted chromatin characteristic of banded regions, but also of non-homologous chromatid regions, which become fused and confluent. Thus, an alternative hypothesis that JIL-1 activity may regulate boundary elements that control the spreading of heterochromatic factors cannot be ruled out, or that the two mechanisms may act in concert. However, the spreading of the dmH3K9 marker and HP1 to ectopic locations on the chromosomes is likely to lead to heterochromatization and repression of gene expression at these sites. The results further suggest the possibility that the lethality of JIL-1 null mutants may be due to the repression of essential genes at these ectopic sites as a consequence of the spreading of Su(var)3-9 activity. This hypothesis is supported by genetic interaction assays that demonstrated that the lethality of a severely hypomorphic JIL-1 heteroallelic combination could be almost completely rescued by a reduction in Su(var)3-9 dosage that prevented the ectopic dimethylation of histone H3K9 (Zhang, 2006).
The Su(var)3-1 alleles of JIL-1 consist of dominant gain-of-function alleles that antagonize the expansion of heterochromatin formation. However, evidence is provided that the underlying molecular mechanism of this antagonism is different from that occurring in the loss-of-function null and hypomorphic JIL-1 alleles described in this study. JIL-1Su(var)3-1 alleles are characterized by deletions of the COOH-terminal domain that do not affect JIL-1 kinase activity or the spreading of heterochromatin markers. Furthermore, the results indicate that the COOH-terminal domain of JIL-1 is required for proper chromosomal localization and that JIL-1Su(var)3-1 proteins are mislocalized to ectopic chromosome sites. Thus, it is proposed that the dominant gain-of-function effect of the JIL-1Su(var)3-1 alleles may be attributable to JIL-1 kinase activity at ectopic locations, possibly through the phosphorylation of novel target proteins, or by mis-regulated localization of the phosphorylated histone H3S10 marker. Although the JIL-1Su(var)3-1 proteins are mislocalized, they still associate with chromosomes and phosphorylate the histone H3S10 residue, suggesting that other regions of the protein have a binding affinity for at least some of the substrates and interaction partners of JIL-1. This is supported by the finding that each of the two kinase domains of JIL-1 can interact with the MSL-complex in vitro (Zhang, 2006).
In summary, evidence is provided that the JIL-1 kinase is a major regulator of histone modifications that affect gene activation, gene silencing and chromatin structure. Thus, it will be informative in future experiments to further explore the interaction of JIL-1 with genes controlling heterochromatin formation, in order to gain a better understanding of the molecular mechanisms of epigenetic gene regulation (Zhang, 2006).
Hypomorphic loss-of-function alleles of the JIL-1 histone H3S10 kinase are strong suppressors of position effect variegation (PEV) of the wm4 allele and lack of JIL-1 activity can counteract the effect of the dominant enhancer E(var)2-1 on PEV. To examine the role that JIL-1 plays in higher-order chromatin structure and gene expression, the effect of an allelic series of hypomorphic JIL-1 alleles on PEV of the wm4 allele was examined. The In(1)wm4 X chromosome contains an inversion that juxtaposes the euchromatic white gene and heterochromatic sequences adjacent to the centromere. The resulting somatic variegation of wm4 expression occurs in clonal patches in the eye reflecting heterochromatic spreading from the inversion breakpoint that silences wm4 expression in the white patches and euchromatic packaging of the w gene in those patches that appear red (Lerach, 2006: full text of article).
Studies of this effect suggest that the degree of spreading may depend on the amount of heterochromatic factors at the breakpoint. In these experiments the In(1)wm4 chromosome was crossed into different JIL-1 mutant backgrounds that combined hypomorphic and null JIL-1 alleles (JIL-1z28, JIL-1z60, and JIL-1z2) to generate progeny expressing different amounts of wild-type JIL-1 protein. The JIL-1z28 allele is a weak hypomorph producing 45% of the normal level of wild-type JIL-1 protein; the JIL-1z60 allele is a strong hypomorph producing only 0.3% of wild-type JIL-1 protein levels, whereas the JIL-1z2 allele is a true null and homozygous animals do not survive to adulthood. The JIL-1h9 allele expresses a truncated JIL-1 protein that lacks part of the second kinase domain and the entire COOH-terminal domain. The JIL-1z2/JIL-1z60 heteroallelic combination is semilethal and only a few eclosed animals from large-scale crosses could be analyzed. Flies with the different genotypes were scored for the percentage of the eye that was red and variegated wm4; +/+ flies containing wild-type levels of JIL-1 protein were used as controls. As JIL-1 protein levels were reduced, an increasing percentage of flies showed fully pigmented eyes, with 100% of the JIL-1z2/JIL-1z60 and JIL-1z2/JIL-1h9 animals showing completely red eyes. This is in contrast to the control crosses in which none of the flies exhibited completely red eyes. These results strongly indicate that loss of JIL-1 protein results in suppression of PEV of the wm4 allele (Lerach, 2006).
To confirm that loss of the JIL-1 protein produces a bona fide Su(var) phenotype, the effect of decreased levels of JIL-1 protein in a wm4 genetic background that also carries the dominant enhancer E(var)2-1 was examined. This enhancer results in nearly completely white-eyed flies and has proven useful in identifying and characterizing strong Su(var) mutations in genetic screens. As levels of JIL-1 protein decreased in this background, a corresponding increase in pigmentation was observed with 51.2% of JIL-1z60/JIL-1z60 and 90.0% of JIL-1z2/JIL-1h9 flies showing completely red eyes compared to 0% in control flies. Thus, lack of JIL-1 activity strongly counteracts the effect of the dominant enhancer E(var)2-1 on PEV of the wm4 allele (Lerach, 2006).
It has been demonstrated that a reduction in the levels of the JIL-1 histone H3S10 kinase results in a redistribution of the major heterochromatin markers H3K9me2 and HP1 to ectopic locations on the chromosome arms with the most pronounced increase on the X chromosomes. Interestingly, overall levels of heterochromatic factors remained unchanged, implying a concomitant reduction in the levels of pericentric heterochromatic factors. On the basis of these findings a model was proposed wherein JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9 mediated heterochromatization. Thus, in the absence of JIL-1 function the dispersion of the H3K9me2 mark and HP1 to ectopic locations on the chromosomes would be expected to lead to heterochromatization and repression of gene expression at these sites, suggesting that loss of JIL-1 would result in an E(var) phenotype if the reporter locus were located at such a site. However, ectopic heterochromatization was not uniform and not all active gene loci are repressed. This implies that certain chromatin sites are molecularly distinct and may be preferentially modified by Su(var)3-9 to recruit HP1 in the absence of JIL-1. Paradoxically, as a consequence of this combined with the redistribution of a fixed level of heterochromatic factors, a reduction in JIL-1 activity would be predicted to lead to suppression—not enhancement—of PEV at loci not affected by ectopic Su(var)3-9 activity but sensitive to the levels of heterochromatic factors at the pericentromeric chromatin, such as has been demonstrated for the wm4 allele. The results of this study support this hypothesis by demonstrating that JIL-1 hypomorphic loss-of-function alleles are strong suppressors of PEV of the wm4 allele and that lack of JIL-1 activity can counteract the effect of the dominant enhancer E(var)2-1 on PEV. It has been proposed that the suppression of PEV of the wm4 allele in JIL-1 hypomorphic backgrounds is due to a reduction in the level of heterochromatic factors at the pericentromeric heterochromatin near the inversion breakpoint site that reduces its potential for heterochromatic spreading and silencing (Lerach, 2006).
It has been demonstrated that the Su(var)3-1 alleles of JIL-1 consist of dominant gain-of-function alleles that also strongly suppress PEV. However, JIL-1Su(var)3-1 alleles are characterized by deletions of the COOH-terminal domain that do not affect JIL-1 kinase activity or the spreading of heterochromatin markers indicated that the COOH-terminal domain of JIL-1 is required for proper chromosomal localization and that JIL-1Su(var)3-1 proteins are mislocalized to ectopic chromosome sites. Thus, the dominant gain-of-function effect of the JIL-1Su(var)3-1 alleles may be attributable to JIL-1 kinase activity at ectopic locations possibly through phosphorylation of novel target proteins or by misregulated localization of the phosphorylated histone H3S10 mark. Consequently, the molecular mechanism of suppression of PEV of the wm4 allele is likely to be different for the dominant gain-of-function Su(var)3-1 alleles and for the hypomorphic loss-of-function JIL-1 alleles (Lerach, 2006).
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date revised: 20 December 2007
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