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 suppressionnot enhancementof 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).
The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. This study addresses the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. The lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene, and Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms is unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity (Deng, 2010).
While Su(var)3-9, Su(var)3-7, and HP1a reciprocal interactions are well documented at pericentric regions they are not universal. For example, HP1 binding on the fourth chromosome has been shown to be independent of Su(var)3-9; a tethering system to recruit HP1a to euchromatic sites has shown that HP1a-mediated silencing can operate in a Su(var)3-9-independent manner. Moreover, evidence has been provided that at least two different molecular mechanisms regulate Su(var)3-9 localization, one dependent on HP1 and one dependent on the JIL-1 kinase. These findings indicate that although Su(var)3-9, Su(var)3-7, and HP1a cooperate in heterochromatin formation and gene silencing at pericentric chromosome sites, they may function independently at other regions such as the chromosome arms. This study shows that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene. This effect was observed with three different alleles of Su(var)3-7, strongly suggesting it is likely to be specific to Su(var)3-7 and not to second site modifiers. Furthermore, evidence is provided that JIL-1 levels and/or activity regulate the chromosome localization of Su(var)3-7 and that Su(var)3-7 levels are dramatically redistributed to the chromosome arms in conjunction with a reduced presence at the chromocenter in the absence of JIL-1 (Deng, 2010).
Previously, it has been demonstrated that JIL-1 genetically interacts with Su(var)3-9 but not with Su(var)2-5, suggesting that the lethality and disruption of chromosome morphology observed when JIL-1 levels are decreased are associated with ectopic Su(var)3-9 activity on the chromosomal arms and unrelated to HP1a recruitment. In this scenario, the spreading of the H3K9me2 mark to ectopic locations on the chromosomes is likely to lead to heterochromatization and repression of gene expression at these sites, leading to increased lethality. This hypothesis has been supported by genetic interaction assays that demonstrate that the lethality of JIL-1 null mutants can be almost completely rescued by a reduction in Su(var)3-9 dosage that prevents ectopic dimethylation of histone H3K9. However, this study has shown that while reducing the dose of Su(var)3-7 also rescues viability of JIL-1 null mutant larvae, H3K9me2 in polytene squashes still spreads to the chromosome arms, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality, but rather that Su(var)3-9-mediated recruitment of Su(var)3-7 is a necessary factor. Futhermore, since viability was rescued despite no obvious improvement in chromosome morphology, the lethality caused by loss of JIL-1 function is not likely to be a consequence of perturbed chromosome morphology. Taken together these observations give rise to a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity. How Su(var)3-7 may mediate these effects is unknown and will require additional studies (Deng, 2010).
The inherent components of heterochromatin Su(var)3-9, HP1a, and Su(var)3-7 display a haplosuppressor/triploenhancer dosage-dependent effect on PEV. Additional copies of all three genes cause strong enhancement of white variegation in wm4, and in genetic interaction tests, the suppressor effect of Su(var)3-9 null mutations dominates the triplo-dependent enhancer effect of Su(var)2-5) and Su(var)3-7. Furthermore, it has been recently demonstrated that the gain-of-function JIL-1Su(var)3-1 allele is one of the strongest suppressors of PEV so far described at all the PEV arrangements that have been tested. This allele even counteracts gene repression that is caused by overexpression of the major determinants of heterochromatin formation, e.g., Su(var)3-9, Su(var)2-5, and Su(var)3-7. The JIL-1Su(var)3-1 allele generates truncated proteins with COOH-terminal deletions that mislocalize to ectopic chromatin sites, leading to expanded histone H3S10 phosphorylation. On the basis of these findings, a model has been proposedfor a dynamic balance between euchromatin and heterochromatin, where as can be monitored in PEV arrangements, the boundary between these two states is determined by antagonistic functions of euchromatic regulators (JIL-1) and the determinants of heterochromatin assembly. This study has further tested this hypothesis using JIL-1 loss-of-function alleles which are shown can act as haploenhancers of PEV. This included PEV of the wm4 allele where, interestingly, combinations of strong hypomorphic JIL-1 alleles act as suppressors, not enhancers. It has been proposed that this is due to a reduction in the levels of heterochromatic factors near the inversion breakpoint that reduces its potential for heterochromatic spreading and silencing. As predicted by this hypothesis it was shown that in chromosome squash preparations from JILz2/+ larvae there was no discernible redistribution of the H3K9me2 heterochromatic mark. It was further demonstrated that JIL-1 and Su(var)3-7 alleles can counteract each other's effect on PEV. In all three PEV arrangements tested, Su(var)3-7 loss-of-function alleles acted as strong haplosuppressors as indicated by a high proportion of nearly completely red eyes, whereas JIL-1 loss-of-function alleles acted as strong haploenhancers as indicated by a high proportion of flies with nearly completely white ommatidia. However, in double mutant backgrounds, variegation of the proportion of red ommatidia was substantively restored and closer to the distribution when wild-type levels of JIL-1 and Su(var)3-7 proteins were present. These results strongly support a genetic interaction between JIL-1 and Su(var)3-7 and provide evidence that a finely tuned balance between the levels of JIL-1 and Su(var)3-7 contributes to the regulation of PEV (Deng, 2010).
While several potential mechanisms for heterochromatin spreading and gene silencing have been identified, the concept of a dynamic balance between euchromatin and heterochromatin implies that euchromatic factors may have similar spreading potential. However, the mechanisms that actively may lead to the expansion of euchromatic domains have received comparatively less attention. In Drosophila, the studies of the JIL-1 kinase have demonstrated that histone H3S10 phosphorylation is an important epigenetic modification potentially regulating both the establishment and maintenance of euchromatin. For example, it has been shown using a LacI tethering system, that JIL-1-mediated ectopic H3S10 phosphorylation can cause a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. Thus, spreading of JIL-1 activity has the potential to expand euchromatic domains and counteract gene silencing in heterochromatic regions. However, while it has been shown that the COOH-terminal region of JIL-1 can directly interact with the histone H3 tail region, it remains to be established how JIL-1 targeting to specific chromatin regions is regulated and how dynamic this regulation is (Deng, 2010).
This study shows that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on position-effect variegation, providing evidence that a finely tuned balance between the levels of JIL-1 and the major heterochromatin components contributes to the regulation of gene expression (Wang, 2011).
The essential JIL-1 histone H3S10 kinase is a major regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, the major heterochromatin markers H3K9me2, HP1a [Su(var)2-5], and Su(var)3-7 spread to ectopic locations on the chromosome arms. These observations suggested a model for a dynamic balance between euchromatin and heterochromatin, where, as can be monitored in position-effect variegation (PEV) arrangements, the boundary between these two states is determined by antagonistic functions of a euchromatic regulator (JIL-1) and the major determinants of heterochromatin assembly, e.g., Su(var)3-9, HP1a, and Su(var)3-7. In support of this model, it has been shown that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on gene expression using PEV assays (Deng, 2010); however, potential dynamic interactions between JIL-1 and the other two heterochromatin genes, Su(var)3-9 and Su(var)2-5, were not addressed previously. Interestingly, in other genetic interaction assays monitoring the lethality as well as the chromosome morphology defects associated with the null JIL-1 phenotype, only a reduction in the dose of the Su(var)3-9 gene rescued both phenotypes. In contrast, in the same assays a reduction of Su(var)3-7 rescued the lethality, but not the chromosome defects (Deng, 2010), and no genetic interactions were detectable between JIL-1 and Su(var)2-5. Thus, these findings indicate that while Su(var)3-9 activity may be a major factor in the lethality and chromatin-structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1a and, to a lesser degree, from Su(var)3-7. This raises the question of whether JIL-1 dynamically interacts with the two other heterochromatin genes, Su(var)2-5 and Su(var)3-9, in regulating gene expression, as it does with Su(var)3-7 (Wang, 2011).
To answer this question, the effect of various combinations of loss-of-function alleles of JIL-1 and Su(var)3-9 or Su(var)2-5 was explored on PEV caused by the P-element insertion line 118E-10. Insertion of this P element (P[hsp26-pt, hsp70-w]) into euchromatic sites results in a uniform red-eye phenotype whereas insertion into a known heterochromatin region of the fourth chromosome results in a variegating eye phenotype. It has been demonstrated that loss-of-function JIL-1 alleles can act as haplo-enhancers of PEV, resulting in increased silencing of gene expression (Deng, 2010), whereas loci for structural components of heterochromatin such as Su(var)3-9, Su(var)2-5, and Su(var)3-7 act as strong haplo-suppressors. In the experiments, the transgenic reporter line 118E-10 was crossed into JIL-1z2/+, Su(var)3-906/+, and Su(var)2-505/+ mutant backgrounds as well as into JIL-1z2/Su(var)3-906 and JIL-1z2/Su(var)2-505 double-mutant backgrounds. The JIL-1z2 allele is a true null allele, the loss-of-function Su(var)3-906 allele is due to a DNA insertion, and the Su(var)2-505 loss-of-function allele is associated with a frameshift resulting in a nonsense peptide containing only the first 10 amino acids of HP1a. Thus, to test whether the heterozygous JIL-1z2 allele could counterbalance the suppression of the Su(var)3-906 or Su(var)2-505 loss-of-function alleles of the PEV of 118E-10, the eye pigment levels of the various genotypes were compared. Pigment assays were performed using three sets of 10 pooled fly heads from each genotype. Although both male and female flies were scored, due to sex differences only results from male flies are shown. However, the trend observed in female flies was identical to that in male flies. The heterozygous JIL-1z2/+ genotype enhances PEV as indicated by the increased proportion of white ommatidia and a 59% decrease in the optical density (OD) of the eye pigment levels. This reduction was statistically significant. In contrast, the heterozygous Su(var)3-906/+ and Su(var)2-505/+ genotypes suppress PEV as indicated by an increase of the proportion of red ommatidia and a statistically significant increase, respectively, in the OD of the eye pigment levels. However, in the JIL-1z2/Su(var)3-906 and JIL-1z2/Su(var)2-505 double-mutant backgrounds, variegation of the proportion of red ommatidia was intermediate, and the eye pigment levels were statistically indistinguishable from genotypes with +/+ levels of JIL-1, Su(var)3-9, and Su(var)2-5 proteins (Wang, 2011).
To test whether a heterozygous JIL-1 null allele also could counterbalance the suppression of the Su(var)3-906 or Su(var)2-505 alleles of the PEV of wm4, experiments were performed similar to those described above for 118E-10. For male flies, the heterozygous JIL-1z2/+ genotype enhances PEV as indicated by a 67% decrease in the OD of the eye pigment levels. This reduction was statistically significant. In contrast, the heterozygous Su(var)3-906/+ and Su(var)2-505/+ genotypes suppress PEV . However, in the JIL-1z2/Su(var)3-906 and JIL-1z2/Su(var)2-505 double-mutant backgrounds, the eye pigment levels were significantly reduced by 13% and 17% as compared to heterozygous Su(var)3-906/+ and Su(var)2-505/+ genotypes, indicating that a heterozygous JIL-1 null allele has the ability to counterbalance the suppression of the Su(var)3-906 or Su(var)2-505 alleles of the PEV of wm4. However, it should be noted that it has been demonstrated that JIL-1 can act both as an enhancer and as a suppressor of wm4 PEV, depending on the precise levels of JIL-1. Thus, the genetic interactions between JIL-1 and the Su(var)3-9 and Su(var)2-5 alleles in regulating the PEV of wm4 are likely to be more complex than in the case of 118E-10 where reduced levels of JIL-1 always act as an enhancer. In females where the enhancer effect of the heterozygous JIL-1z2 allele is less pronounced than in males, a statistically significant counterbalancing effect was detected only in flies of the JIL-1z2/Su(var)2-505 genotype (Wang, 2011).
These results demonstrate that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on the PEV of two different alleles. In previous experiments, a genetic interaction between JIL-1 and Su(var)2-5 was not detected. However, the assays used to probe for interactions were viability and rescue of polytene chromosome morphology. As indicated by the experiments presented in this study, these parameters are likely to be independent of and separate from the mechanisms contributing to epigenetic regulation of PEV and gene silencing. Consequently, the present experiments, taken together with those of Deng, (2010) using a JIL-1 null allele, provide strong evidence that a finely tuned balance between the levels of JIL-1 and all of the major heterochromatin components Su(var)3-9, HP1a, and Su(var)3-7 contributes to the regulation of PEV and gene expression (Wang, 2011).
Drosophila telomere maintenance depends on the transposition of the specialized retrotransposons HeT-A, TART, and TAHRE. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. This study describes two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of Drosophila telomeres, the HeT-A retrotransposon, thus guaranteeing genome stability. Mutant alleles of JIL-1 were found to have decreased HeT-A transcription, putting forward this kinase as the first positive regulator of telomere transcription in Drosophila described to date. The decrease in HeT-A transcription in JIL-1 alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the HeT-A promoter. Moreover, Z4 mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at Drosophila telomeres. Interestingly, a biochemical interaction was detected between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, it is demonstrated that a phenotype of telomere instability similar to that observed for Z4 mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. A model is proposed to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of Drosophila telomeres, such as HP1a (Silva-Sousa, 2012).
Although in Drosophila the role of JIL-1 in activating transcription has remained controversial, at least in the HeT-A, TART, and TAHRE (HTT) array it could act as a positive regulator of transcription for three different reasons: 1) When telomere elongation is needed, a fast activation of HeT-A transcription should be expected. Accordingly, the mammalian JIL-1 orthologous MSK1/2 have been shown to rapidly induce gene expression on the face of stress or steroid response. 2) HeT-A is embedded into the HTT array, a domain that needs to be protected from the influence of the repressive heterochromatin of the neighboring TAS domain. JIL-1 has been suggested to protect the open chromatin state from the spreading of neighboring repressive chromatin at certain genomic positions. 3) The decrease in expression that was observed in the JIL-1 mutants is moderate. Recent data at genomic level revealed that JIL-1 function agrees with a reinforcement of the transcriptional capability of a particular genomic domain rather than net activation (Silva-Sousa, 2012).
Phalke (2009) suggest that JIL-1 has a role in retrotransposon silencing in general and has no effect on telomere transcription. A possible explanation for this discordance with the current results and hypothesis is that the mutant allele of JIL-1 assayed by Phalke, the JIL-1Su(var)3-1 allele, corresponds to a C-terminal deletion of the JIL-1 protein that causes the protein to miss-localize and phosphorylate ectopic sites. The ectopic phosphorylation caused by the JIL-1Su(var)3-1 allele would activate the expression above wild type levels in those genes that normally are not targeted by JIL-1, as it happens to be the case for the Invader4 retrotransposon. The current study has assayed the JIL-1Su(var)3-1 allele obtaining similar result than for the wild type stock, likely for similar reasons. Supporting this, in addition of the JIL-1Su(var)3-1, data from two more JIL-1 alleles, JIL-1z60 and JIL-1z2, is presented that correspond to loss of function alleles and, in both cases, result in a substantial decrease in HeT-A transcription. Moreover, the changes in telomere transcription reported in this study have been assayed directly on the major component of the HTT array, and not through a reporter. The current data demonstrates that JIL-1 is necessary to maintain active transcription of the telomeric retrotransposon HeT-A or, what is the same, transcription from the telomeres in Drosophila (Silva-Sousa, 2012).
Although it was demonstrated that JIL-1 is necessary to maintain transcription from the HTT array, no decrease was detected in telomere length in the JIL-1 mutant alleles. A reasonable explanation for this observation is that the JIL-1 mutant alleles here analyzed (JIL-1z60 and JIL-1z2) have been maintained as heterozygous. It is therefore possible that one copy of JIL-1 is enough to promote enough HeT-A transcription to elongate significantly the telomeres when needed (Silva-Sousa, 2012).
Although in the case of the hypomorph mutation Z47.1 an increase was observed in HeT-A transcription and HeT-A copy number significantly above the control strain (w1118), the null alleles Z42.1 and pzg66 do not show an up-regulation of HeT-A transcription or an increase in its copy number. Although all the stocks were crossed to the w1118 strain to minimize the effects of the genetic background, it could still have a certain influence when comparing the pzg66 allele with the Z47.1. Nevertheless the Z47.1 and Z42.1 alleles come from the same genetic background. A possible explanation could rely on the fact that the Z47.1 mutation is a hypomorph mutation where a small amount of Z4 protein is still present. By ChIP analyses an increase of JIL-1 protein was detected at the HeT-A promoter above control levels, which could explain in part the major transcription of HeT-A in this mutant background, it is possible that although low, the amount of Z4 present in the Z47.1 allele is enough to recruit JIL-1 to the HeT-A promoter. In the pzg66 and the Z42.1 null alleles, JIL-1 cannot be recruited towards the HeT-A promoter and there is no increase in transcription. Nevertheless, with the current data it cannot be concluded that Z4 directly controls the level of HeT-A transcription (Silva-Sousa, 2012).
A phenotype of telomere instability was detected in all three Z4 mutant alleles Z47.1, Z42.1 and pzg66, suggesting a role of this chromosomal protein in guaranteeing telomere stability in Drosophila. Although a number of genes involved in the capping function in Drosophila still remain unidentified, there is no evidence that Z4 directly participates in the protection of the telomeres. Mutant alleles of genes directly involved in the capping function, such as woc or caravaggio (HOAP), show multiple and more numerous TFs in larval neuroblasts than the ones that were observed in the Z4 mutant alleles. Moreover, it has been possible to detect staining for one of the major capping components, the HOAP protein, in the TFs of Z4 mutant neuroblast cells, indicating that the telomere-capping complex is still loaded to a certain degree. Instead of directly participating in the capping, it is hypothesized that the major chromatin changes caused by the lack of Z4 at the HTT array result in a secondary loss of necessary chromatin and capping components like HP1a (Silva-Sousa, 2012).
Results from the ChIP experiments suggest a relationship between JIL-1, Z4 and HP1a in fine-tuning the chromatin structure at the HTT array. HP1a has a dual role at the telomeres explained by its participation in both the capping function and the repression of gene expression that also exerts in other genomic domains. In the HP1a Su(var)2-505 allele, which it is known to have a major transcription of HeT-A and problems of telomere stability, a pronounced decrease was observed in Z4 and JIL-1. In the Z47.1 allele the decrease in Z4 protein is accompanied by a similar decrease in H3K9me3 and HP1a at the HeT-A promoter. Finally in the JIL-1z60 allele the increase in silencing epigenetic marks like H3K9me3 and HP1a is also accompanied by a decrease in Z4. In particular, the pronounced dependence of the presence of HP1a and Z4, points toward the loss of HP1a and H3K9me3 to a possible cause for telomere instability in the Z4 mutant alleles here studied. Interestingly, in the Su(var)2-504/Su(var)2-505 heteroallelic combination (considered a null mutation), 15% of telomeres involved in telomere associations are still able to recruit the HOAP protein. Therefore the data on HOAP localization in the Z4 mutant alleles is still consistent with the TFs being caused by the decreased availability of HP1a in these cells. The above results demonstrate that Z4 in a coordinated manner together with JIL-1 and HP1a is an important component of the telomere chromatin in Drosophila, which upon its reduction causes significant changes in the chromatin of the HTT array, which are the cause of the observed telomere instability in all the Z4 mutant alleles here studied (Silva-Sousa, 2012).
It has been possible to detect a biochemical interaction between JIL-1 and Z4, and the data suggests that these two proteins can be components of the same protein complex. This interaction had been previously suggested because both proteins have been found co-localizing in different genomic locations, but no direct proof existed to date. In each genomic location where the Z4-JIL-1 complex is needed, a special mechanism of recruitment should exist. Importantly, it has been shown how Z4 specifically interacts with HeT-A Gag. HeT-A Gag is the only protein encoded by the HeT-A element and has been shown to specifically localize at the telomeres. HeT-A Gag has been shown to be in charge of the targeting of the transposition intermediates for the HeT-A element and also for its telomeric partner the TART retrotransposon. Interestingly, when the consequences for telomere stability were studied after knocking down the HeT-A gag gene by RNAi, similar TFs were observed than when knocking down the Z4 gene, further relating the action of both genes in telomere stability. Z4 is known to participate in different protein complexes with roles in different genomic locations. Because it has been demonstrated that Z4 is able to associate with a variety of proteins in these complexes, it is thought that the description of a mechanism for its specific targeting to telomeres through one of the telomeric retrotransposon proteins is especially relevant (Silva-Sousa, 2012).
Integrating information from previous literature and the results exposed by this study, a possible model to describe the state of the chromatin at the HTT array in each of these three mutant scenarios; JIL-1, Z4 and Su(var)2-5, as well as in wild type (see Model of the chromatin environment at the HeT-A promoter). The following phenotypes are proposed: (A) Wild type: Z4 defines a boundary at HeT-A promoter that protects from the action of HP1a and other heterochromatin markers. JIL-1 guarantees a certain level of euchromatin inside the HeT-A promoter in order to allow gene expression, (B) JIL-1 mutants: destabilization of the Z4 boundary and the heterochromatin spreads into the HeT-A promoter (enrichment in HP1 and H3K9me3), (C) Z4 mutants: Disappearance of the Z4 boundary, increase in euchromatin marks (H3K4me3) and decrease in heterochromatin marks (HP1a and H3K9me3). Subtle increase in JIL-1 and in euchromatinization of the HeT-A promoter, (D) In Su(var)2-5 mutants: The lack of HP1a allows relaxation of the Z4 boundary causing a JIL-1 and Z4 spread along the HTT array and a relative decrease of these proteins inside the HeT-A promoter. Although the levels of JIL-1 inside the HeT-A promoter are lower than in wild type, the release of silencing caused by loss of HP1a results in increased HeT-A expression (Silva-Sousa, 2012).
It should be taken into account that 1) HP1a has been shown to spread along the HeT-A sequence. 2) The structure and the phenotypes of the different Z4 mutant alleles suggest a possible role of this protein in setting and maintaining the boundaries between heterochromatin and euchromatin in polytene chromosomes. 3) JIL-1 has been extensively shown to be important to counteract heterochromatinization and, when missing, causes a spreading of heterochromatin markers such as H3K9me2, HP1a and Su(var)3-7. 4) JIL-1 has been found to co-localize with Z4 at the band-inter-band transition in polytene chromosomes and also to co-purify with Z4 in different protein complexes. In addition to this, it has been possible to detect a biochemical interaction between JIL-1 and Z4, as well as, a certain dependence on the presence of JIL-1 for the proper localization of Z4, suggesting a possible role of JIL-1 upstream of Z4. Finally, 5) The ChIP analyses in this study suggest a certain dependence of Z4 on HP1a or onto similar chromatin requirements for the loading of both proteins at the HTT array, more specifically at the HeT-A promoter. Summarizing all of the above, it is proposed that the chromatin at the HeT-A promoter could have the following structure: In a wild type situation, the HeT-A promoter contains intermediate levels of HP1a, JIL-1 and Z4. HP1a would be spread along the HTT array, JIL-1 would be concentrated at the promoter region of HeT-A guaranteeing certain level of expression and Z4 would be important to set the boundary between these two opposite modulators (Silva-Sousa, 2012).
In a JIL-1 mutant, the lack of JIL-1 would disturb the Z4 boundary causing a slight decrease in the Z4 presence. This result is in agreement with a Z4-JIL-1 partial interaction. The decrease in JIL-1 presence and the disturbance of the boundary causes a spreading of HP1a into the HeT-A promoter, increasing its presence and repressing transcription from the HTT array (Silva-Sousa, 2012).
In a Z4 mutant, the disappearance of the boundary together with the significant decrease in H3K9me3 causes a decrease in HP1a binding and a substantial modification of the chromatin at the HTT array. The lack of sufficient HP1a at the HTT array causes a destabilization of the chromatin at the cap domain triggering telomere instability as a result. This scenario applies to the three Z4 mutant alleles present in this study, the hypomorph Z47.1, and the nulls pzg66 and Z42.1. On one hand the loss of some Z4 in Z47.1/Z47.1 genotype produces overexpression of HeT-A because in addition to a relaxation of the chromatin, part of JIL-1 is still recruited to the HeT-A promoter and activates transcription in a more effective way than in a wild type situation (Silva-Sousa, 2012).
Finally, in a Su(var)2-5 mutant background, the lack of HP1a along the HeT-A sequence allows a relaxation of the boundary causing a spread of JIL-1 and Z4 from the HeT-A promoter towards the rest of the array and creating as a consequence, permissive chromatin environment releasing HeT-A silencing (Silva-Sousa, 2012).
The model does not completely explain the complex relationships that regulate telomere chromatin, likely because other important components are yet to be described or associated with the ones presented in this study. For example, other chromatin regulatory components that have been associated with Drosophila telomeres included the deacetylase Rpd3, with a regulatory role on chromatin structure, and the histone methyltransferase SetDB1 and the DNA methylase Dnmt2 which by acting in the same epigenetic pathway repress transcription of HeT-A as well as of retroelements in general. Future in depth studies on additional chromatin components will allow completion and detailing even more the description of the chromatin at the HTT array, and allow a better understanding of the mechanism of retrotransposon telomere maintenance and the epigenetic regulation of eukaryote telomeres in general. In the meantime, this study describes a plausible scenario in the view of the transcription and ChIP data (Silva-Sousa, 2012).
The results shown in this study demonstrate the role of JIL-1 as the first described positive regulator of telomere (i.e. HeT-A) expression in Drosophila. Because HeT-A is in charge of telomere maintenance in Drosophila, these results are key to understand how telomere elongation is achieved in retrotransposon telomeres. It was also demonstrated that Z4 is necessary to guarantee telomere stability. The data presented in this study strongly suggest that JIL-1 and Z4 exert these functions by maintaining an appropriate telomere chromatin structure by a coordinated action together with other known telomere components such as HP1a. Moreover, this study shows that JIL-1 and Z4 interact biochemically. Last, and importantly for understanding how the specific role of the Z4-JIL-1 complex at the telomeres is defined and differentiated from its role in other genomic regions, it was shown that Z4 might interact with the HeT-A Gag protein, providing evidence for a targeting mechanism that specifically retrieves this complex to the telomeres (Silva-Sousa, 2012).
In Drosophila, the non-LTR retrotransposons HeT-A, TART and TAHRE build a head-to-tail array of repetitions that constitute the telomere domain by targeted transposition at the end of the chromosome whenever needed. As a consequence, Drosophila telomeres have the peculiarity to harbor the genes in charge of telomere elongation. Understanding telomere expression is important in Drosophila since telomere homeostasis depends in part on the expression of this genomic compartment. Recent studies have shown that the essential kinase JIL-1 is the first positive regulator of the telomere retrotransposons. JIL-1 mediates chromatin changes at the promoter of the HeT-A retrotransposon that are necessary to obtain wild type levels of expression of these telomere transposons. The present study shows how JIL-1 is also needed for the expression of a reporter gene embedded in the telomere domain. This analysis, using different reporter lines from the telomere and subtelomere domains of different chromosomes, indicates that JIL-1 likely acts protecting the telomere domain from the spreading of repressive chromatin from the adjacent subtelomere domain. Moreover, the analysis of the 4R telomere suggests a slightly different chromatin structure at this telomere. In summary, these results strongly suggest that the action of JIL-1 depends on which telomere domain, which chromosome and which promoter is embedded in the telomere chromatin (Silva-Sousa, 2013).
Both the experiments of telomere position effect on the mini-white insertions and the real-time PCR quantifications of the expression of the different telomeric elements, indicate that the lower amount of JIL-1 present in the mutant alleles JIL-1z60 and JIL-1h9 assayed in this study results in a decreased expression of the HTT array. These experiments, in accordance with recent published data, confirm that JIL-1 is necessary to obtain wild type levels of gene expression from the HTT array (Silva-Sousa, 2013).
Additionally, these experiments have also revealed that the effect of JIL-1 is stronger over the HeT-A promoter than over the mini-white promoter existent in the reporter lines used in this study. Similarly, when the effect was assayed of mutations of the Su(var)2-5 gene, known to greatly de-repress the expression of the HeT-A retrotransposon, only a faint de-repression was obtained of the mini-white gene of the lines EY08176, EY09966 and EY00453. In agreement with these observations, another study found similar results using two additional Su(var)2-5 mutant alleles, Su(var)2-502 and Su(var)2-504, over the same reporter lines (Silva-Sousa, 2013).
In summary, JIL-1 and HP1a control gene expression from the telomere domain in Drosophila, being the HeT-A promoter especially sensitive to their effect (Silva-Sousa, 2013).
Although JIL-1 has not been found to localize at the subtelomere domain, TAS, the observation that gene expression varies in this domain in a JIL-1 mutant background, suggests a role of this protein related with a putative boundary between the telomere and subtelomere domains. A pronounced increase was observed in mini-white expression at the TAS domains from the 2L and 2R telomeres when placed in a JIL-1 trans-heterozygous (JIL-1z60/JIL-1h9) mutant background. Accordingly, the ChIP data reveals a significant decrease of H3K27me3 upstream of the mini-white insertion when a JIL-1 trans-heterozygous background is present. Integrating these results, a model is proposed in which the JIL-1 kinase acts as a boundary protecting the promoters of the HTT retrotransposons from the highly compacted chromatin of the adjacent TAS domain. This model is in agreement with an increase of repressive chromatin at the HTT array in JIL-1 mutations. Moreover, the model also reflects the previously reported role of JIL-1 in the protection of the excessive spreading of heterochromatin to adjacent domains, and suggests that JIL-1 could exert a barrier function at the HTT-TAS boundary, based on the results obtained from this study (Silva-Sousa, 2013).
A different behavior was not observedin the control of gene expression when studying the HTT array of the 4th and the 2nd chromosomes. Interestingly, a significant difference was found when looking at the subtelomere of the 4R telomere. In the reporter lines 39C-72 and 118E-5 from the subtelomere domain of the 4th chromosome, it was found that JIL-1 is important to allow gene expression and HP1a is necessary to repress it. This finding indicates that the chromatin of the subtelomere domain in the 4th chromosome is not equivalent to the TAS chromatin in the other chromosomes (Silva-Sousa, 2013).
In contrast, the results indicate that the chromatin at the 4R subtelomere of the reporter lines used in this study is less compacted and more permissive to gene expression than the TAS domains from the other chromosomes, and even than the HTT array. This scenario suggests that in the 4R telomere of these lines the compaction of the chromatin is in the opposite orientation than in the rest of the telomeres. It would be interesting to study if this reversed chromatin organization with respect to the other telomeres has a particular role in the general telomere function in Drosophila (Silva-Sousa, 2013).
This study has reported how the presence of the JIL-1 kinase is needed at the telomere in Drosophila in order to allow gene expression of the promoters embedded in this domain. The lack of JIL-1 causes an increase of silencing at the HTT array and a release of silencing at the subtelomeric domain likely by the spreading of heterochromatin towards adjacent domains. Finally, it was discovered that the telomere and subtelomere domain of the 4R arm of some lines might have a different chromatin organization with respect to the other Drosophila telomeres (Silva-Sousa, 2013).
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date revised: 10 February 2014
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