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Suppressor of variegation 3-3: Biological Overview | References

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified (Shi, 2004). Lsd1 is highly conserved, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3 (Shi, 2004; Metzger, 2005). This study, along with a second study by Rudolph (2007) describes the effects of Lsd1 [Su(var)3-3] mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development (Di Stefano, 2007).

Su(var)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 dictates the distinction between euchromatic and heterochromatic domains during early embryogenesis. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. Su(var)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. These data indicate an early developmental function for the Su(var)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which Su(var)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation (Rudolph, 2007).

Originally, Lsd1 was found as a component of corepressor complexes (Hakimi, 2002; Humphrey, 2001; Shi, 2003; You, 2001; Ballas, 2001). Lsd1-demethylase activity was only discovered recently (Shi, 2004) and was found to be modulated by its associated proteins, such as CoREST (Lee, 2005). Lsd1 depletion in mammalian cells correlates with increased gene expression and elevated levels of H3-K4 methylation at target promoters (Shi, 2004). However, Lsd1 can also act as a coactivator and demethylates H3-K9, a repressive mark (Metzger, 2006; Di Stefano, 2007 and references therein).

Lsd1 is evolutionary conserved (Shi, 2004), but little is known about its biological function. To address this question, flies were generated carrying a mutation in the sole Drosophila gene that exhibits high homology to Lsd1, CG17149/dLsd1. dLsd1 contains both a putative amine-oxidase domain and a SWIRM domain. In the Exelixis collection of mutants, two piggyBac insertions were found in the vicinity of CG17149/dLsd1: f03544 (designated as dLsd1¹) and f00678 (dLsd1²). Using Flp-recombination target (FRT) sites in the piggyBac transposon to promote trans-recombination between dLsd1¹ and dLsd1², a deletion allele of dLsd1, dLsd1^ΔN was generated (Di Stefano, 2007).

Southern-blot analysis confirmed the authenticity of the dLsd1 alleles. dLsd1^ΔN lacks the presumptive promoter region and the N-terminal portion of the gene, including the SWIRM domain. Quantitative PCR analysis with primers specific for the 5′ end of dLsd1 confirmed the absence of these sequences in dLsd1^ΔN homozygous flies. Low levels (<20%) of 3′ transcripts persist in the mutant animals, but any potential products would lack the putative nuclear-localization signal and the SWIRM domain and are unlikely to be functional. The SWIRM domain is thought to function in protein–protein interactions, DNA–protein interactions, and enzyme catalysis. Inactivation of this domain greatly reduces the stability and demethylase activity of Lsd1 (Chen, 2006; Stavropoulos, 2006). Western-blot analysis showed that dLsd1 is expressed at high levels in wild-type (WT) flies, but no dLsd1 protein was detected in dLsd1^ΔN-homozygous flies. Hence dLsd1^ΔN is, most likely, a null allele (Di Stefano, 2007).

This collection of mutant alleles provided an opportunity to study the biological function of dLsd1 in Drosophila. First, the effects of dLsd1 mutation on viability was assessed. Crosses of dLsd1^ΔN-heterozygous animals gave only one-third of the expected number of dLsd1^ΔN-homozygous progeny. Interestingly, this reduction in viability is more dramatic in the male progeny (approximately 90% of the viable dLsd1^ΔN homozygotes were females) (Di Stefano, 2007).

dLsd1^ΔN mutants are sterile. In these animals, ovary development is severely impaired. The Drosophila ovary consists of approximately 16 ovarioles, which are chains of developing egg chambers with a germarium at the anterior tip. The germarium contains germline stem cells (GSC) and somatic stem cells (SSC), which give rise respectively to the germline cysts and to follicle cells. Interestingly, DNA staining shows that dLsd^ΔN mutant ovaries lack proper ovariole structures and that the formation of egg chambers is abnormal at very early stages. Both the germline and follicle cells appear abnormal and, strikingly, follicle cells fail to properly encapsulate the 16-cell cysts. In males, the testes are morphologically intact, but DNA staining suggests defects during spermatogenesis. Interestingly, dLsd1^ΔN homozygotes also have a held-out-wing phenotype that renders them unable to fly (Di Stefano, 2007).

To confirm that these defects are due specifically to dLsd1 loss and are not the result of secondary mutations, complementation tests were performed with a deficiency [Df(3L)ED4858] that uncovers the dLsd1 gene. Trans-heterozygotes carrying dLsd1^ΔN and Df(3L)ED4858 recapitulated the phenotypes observed in dLsd1^ΔN-homozygous flies (Di Stefano, 2007).

It is concluded that dLsd1 mutation reduces viability in a gender-dependent manner, causes abnormal ovary development, and results in animal sterility. Collectively, these results point to important roles for dLsd1 in the late stages of development. dLsd1 levels are highest in the embryonic stages, suggesting that dLsd1 might also have a function during early stages of development that may be masked in the dLsd1-homozygous mutants by maternally supplied products (Di Stefano, 2007).

Biochemical studies with human Lsd1 have led to reports that Lsd1 can act both as a corepressor of transcription by demethylating H3-K4 (Shi, 2004) and as a coactivator by demethylating H3-K9 (Metzger, 2005). To determine which of these potential activities is predominant in Drosophila, the levels of histone modification were examined in dLsd1 mutant flies. The global level of monomethyl and dimethyl H3-K4 was considerably higher in dLsd1 mutants than in wild-type flies; this effect was particularly striking in adult males. In contrast, no increase in the global levels of methyl H3-K9 was found; indeed the level of dimethyl and trimethyl H3-K9 decreased slightly in dLsd1 mutants. The levels of monomethyl H4-K20, dimethyl H3-K27, dimethyl H4-K20, trimethyl H3-K36, and acetyl H3-K9 were unaltered in dLsd1^ΔN mutant flies. Interestingly, ovaries contain a higher level of dLsd1 and a lower level of monomethyl H3-K4 than does the rest of the adult female, potentially explaining why dLsd1 mutation selectively perturbs ovary development (Di Stefano, 2007).

As a further test, pUAST-dLsd1 transgenic flies were generated, and it was found that increased levels of dLsd1 reduced dimethyl and monomethyl H3-K4, confirming that dLsd1 is a critical determinant of the global level of H3-K4 methylation. Surprisingly, these animals lacked any clear developmental defects (Di Stefano, 2007).

H3-K4-methyl residues are highly enriched in euchromatin. To test whether the elevated levels of monomethyl and dimethyl H3-K4 in dLsd1 mutants alter the balance between euchromatin and heterochromatin, three variegating systems [T(2;3)Stubble^variegated (Sb^v), In(1)y^3Pyellow, and In(1)white^m4h] were used, and it was asked whether dLsd1 alleles modify positional-effect variegation (PEV). PEV is the mosaic pattern of gene silencing observed when genes that are normally located in euchromatin regions are placed into a heterochromatic environment. Suppressors of PEV include mutants of heterochromatin-associated proteins, such as HP1, and the histone H3-K9 methyltransferase Su(var)3-9 (Di Stefano, 2007).

T(2;3)Sb^v translocation juxtaposes the Sb mutation and the centric heterochromatin of the second chromosome, resulting in mosaic flies with both Sb and normal bristles. Activation of dominant Sb results in stubble bristles. When T(2;3)Sb^v was crossed to dLsd1^ΔN, a significant increase was observed in the frequency of Sb bristles. Similar results were found with the yellow locus. Analysis of bristles in the wing of In(1)y^3P/+; +; dLsd1^ΔN/+ flies showed that a single allele of dLsd1^ΔN or dLsd1² suppressed yellow variegation, resulting in a 25% reduction of yellow bristles. Suppression of variegation was also observed with w^m4h. These results indicate that dLsd1 alleles are potent suppressors of PEV and suggest that the absence of dLsd1 alters chromatin structure (Di Stefano, 2007).

Monomethyl and dimethyl forms of H3-K4 are linked to transcriptional activation; the increased level of these modifications in dLsd1^ΔN mutants suggests that dLsd1 normally represses transcription. Previous studies with human cells have shown that Lsd1 regulates the expression of neuron-specific genes (Shi, 2004). To determine whether this function is conserved, RNAi was used to deplete dLsd1 from SL2 cells, and the expression of neuron-specific genes, such as Nicotinic Acetylcholine Receptor β (nAcrβ) and Na channel (Nach), was examined. dLsd1 depletion increased the level of monomethyl H3-K4 and increased the expression of both nAcrβ and Nach, indicating that Lsd1's role in the repression of neuron-specific genes is conserved between Drosophila and humans (Di Stefano, 2007).

The homeobox (Hox) gene locus is subject to extensive H3-K4 methylation by trithorax-group proteins. It was therefore asked whether the expression level of the Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) is affected by dLsd1 depletion. Ubx- and abdA-mRNA levels increased 2-fold in SL2 cells treated with dLsd1 double-stranded RNA (dsRNA). These changes were specific and were not seen with other control genes (dDP and Hid). To verify the relevance of these observations in vivo, the expression of these genes was compared in wild-type and dLsd1^ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1^ΔN mutant flies, confirming the importance of dLsd1-mediated repression in vivo. Intriguingly, it was observe that this upregulation is age dependent: The difference in gene expression is minimal in larval stages, and, consistent with this, the Hox gene-expression pattern in imaginal discs from dLsd1^ΔN mutant larvae and in embryos is largely unaltered. However, the level of nAcrβ, Ubx, and Abd-B gradually and significantly increases with age after eclosion, suggesting that dLsd1 function is especially important for the regulation of gene expression in adult tissues (Di Stefano, 2007).

Using Drosophila, this study has examined the consequences of eliminating Lsd1 function. The results help to place the previously described biochemical activities of Lsd1 into a biological context and show that dLsd1 is a key determinant of the global levels of monomethyl and dimethyl H3-K4 in vivo. dLsd1 mutation impacts heterochromatin homeostasis and leads to ectopic expression of a subset of genes in flies. Curiously, it was observed that the consequences of the global upregulation of H3-K4 methylation on animal development are restricted to a few specific organs. One of the clearest defects is in the ovary, where dLsd1^ΔN mutant egg chambers are abnormal early in development and follicle cells fail to encapsulate the cyst. This defect is consistent with the observation that dLsd1 is highly expressed in the ovary, and indicates that dLsd1-mediated demethylation of H3-K4 plays a crucial role in this organ. It is suggested that, in dLsd1^ΔN mutants, the elevated expression level of dLsd1 target genes causes tissue-specific defects. Hox genes and neuron-specific genes are among the targets upregulated in dLsd1^ΔN mutants. The upregulation occurs late in development, and this may explain the lack of homeotic phenotypes or other early developmental defects in dLsd1^ΔN mutants. The stronger changes in histone modification seen in dLsd1^ΔN mutant males compared to females and the sex-dependent lethality caused by dLsd1 mutation raise the possibility that dLsd1 may have a sex-specific distribution on chromatin; alternatively, its mutation may alter the chromatin distribution of the male-specific lethal complex (Di Stefano, 2007).

Whereas mammalian Lsd1 has been shown to be able to demethylate H3-K9, no increase was seen in the global level of dimethylated H3-K9. This might indicate that this activity is not conserved in Drosophila or that it is redundant with other demethylases; alternatively, it might be restricted to specific loci and/or specific tissues (Di Stefano, 2007).

This study opens the road to further studies aimed at delineating the specific functions of dLsd1 in the control of gene expression. Genome-wide studies will be necessary to identify all the dLsd1-regulated genes and to identify the target genes responsible for each of the developmental defects (Di Stefano, 2007).

One important implication of the tissue-specific defects seen in the dLsd1^ΔN mutant animals is the possibility that dLsd1 may be manipulated in vivo to modulate specific biological processes. The results support the idea that global changes in the levels of histone methylation can impact specific developmental processes. These results also highlight the need for additional studies to understand how histone methylation is dynamically regulated in vivo and how these changes contribute to normal development and disease (Di Stefano, 2007).

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

Su(var)3-3 mutations belong to the strongest dominant suppressors for white gene silencing in the w^m4 PEV rearrangement (Wustmann, 1989). Su(var)3-3 mutations are characterized by a number of interesting phenotypic effects. Homozygous mutant females produce no oocytes (agametic), whereas in males only immobile sperms are found (Szabad, 1988). These mutations are also butyrate sensitive and display recessive lethal interactions with additional heterochromatin, such as an extra copy of the Y chromosome in XXY females or in XYY males (Rudolph, 2007).

All 22 Su(var)3-3 mutations were isolated as dominant suppressors of white variegation in w^m4, and 15 alleles displayed mutational lesions in the protein-coding sequences. In the remaining seven alleles, no transcript is detected by RT-PCR. Phenotypic rescue of the dominant suppressor effect of Su(var)3-3 heterozygotes is restored by an extra gene copy of a wild-type allele of Su(var)3-3. Genetic crosses demonstrate that the Su(var)3-3 mutations also strongly suppress heterochromatic PEV in other rearrangements, such as brown, Stubble, yellow, or a lacZ transgene. The general suppressor effect of Su(var)3-3 mutations in PEV illustrates its crucial requirement for heterochromatic gene silencing (Rudolph, 2007).

The data support a model in which heterochromatin formation and gene silencing in PEV are defined during early embryonic development of Drosophila. A dynamic balance between HMTases and demethylases controls establishment of the functionally antagonistic histone H3K4 and H3K9 methylation marks at the border region of euchromatin and heterochromatin. In transcriptionally silent cleavage nuclei, chromatin is in a naive state with only little H3K9me2 and with H3K4 methylation completely missing. A dramatic transition of chromatin structure occurs during blastoderm formation and cellularization by establishing H3K4 and H3K9 methylation. In contrast to H3K9 acetylation, which is already found in cleavage chromatin, H3K4 methylation at prospective euchromatin appears first at the end of cleavage in cycle 12. In parallel, di- and trimethylation of H3K9 and HP1 binding establish heterochromatin. Pole cells, which are the primordial germ cells of Drosophila, are in a transcriptionally silent state and show extensive H3K9me2 and H3K9me3. During the definition of the euchromatin-heterochromatin boundaries in blastoderm cells and for the establishment of repressive H3K9 methylation marks in primordial germ cells, the SU(VAR)3-3 demethylase plays an early and inductive regulatory role. SU(VAR)3-3 might also be involved in control of early transcriptional activities within Drosophila pericentromeric sequences preceding heterochromatin formation, as suggested by a model of heterochromatin formation that depends on the RNAi pathway (Rudolph, 2007).

Genetic analysis revealed that SU(VAR)3-3 functions upstream of the H3K9 HMTase SU(VAR)3-9 and the heterochromatin-associated proteins HP1 and SU(VAR)3-7 in control of gene silencing in PEV. Combined with earlier studies of epigenetic interactions, heterochromatic gene silencing is established by a sequential action of SU(VAR)3-3, SU(VAR)3-9, the amount of Y heterochromatin, HP1, and SU(VAR)3-7. RPD3 also acts upstream of SU(VAR)3-9, because Rpd3 mutations dominate the dose-dependent PEV enhancer effect of SU(VAR)3-9. Additional genomic copies of Su(var)3-3 are epistatic to a Rpd3 mutation placing the H3K4 demethylase SU(VAR)3-3 together with RPD3 at the top of a mechanistic hierarchy controlling heterochromatic gene silencing in Drosophila. Such a role is in agreement with the enriched association of SU(VAR)3-3 to prospective heterochromatin in early blastoderm nuclei. In Su(var)3-3 null embryos, there is an extension of H3K4me2 and concomitant reduction of H3K9me3 at prospective heterochromatin, suggesting that SU(VAR)3-3 has a protective function at heterochromatic regions to restrict expansion of H3K4 methylation. Similarly, H3K9 acetylation becomes expanded toward heterochromatin. H3K4 methylation precedes H3K9 methylation in blastoderm nuclei, and both SU(VAR)3-3 and SU(VAR)3-9 are abundant proteins within cleavage chromatin. A developmentally regulated silencing complex between SU(VAR)3-3, RPD3, and SU(VAR)3-9 is therefore likely to dictate the distinction between euchromatic and heterochromatic domains during early embryogenesis. A comparable functional crosstalk between human LSD1 and HDAC1/2, which depends on nucleosomal substrates and the CoREST protein, has been demonstrated in vertebrates (Lee, 2006). The interaction between SU(VAR)3-3 and RPD3 could also explain butyrate sensitivity of Su(var)3-3 mutations (Reuter, 1982). The effect of SU(VAR)3-3 on heterochromatin formation during blastoderm could involve both maternal and zygotic protein. Association of SU(VAR)3-3 with cleavage chromatin is dependent on maternal sources. In contrast, all other effects on gene silencing are zygotically determined, and no maternal effects on PEV were found in any of the Su(var)3-3 mutations. This is also supported by clonal analysis showing early onset and stable maintenance of gene silencing in PEV (Rudolph, 2007).

Transcriptional silence in primordial germline cells of Drosophila is regulated by nanos (nos), pumillio (pum), and germ cell-less (gcl), since mutations in these genes result in premature activation of transcription in germ cells. However, in nos null embryos, only about 50% display H3K4 methylation signals, suggesting that several other factors contribute to establishment and maintenance of transcriptional silence. SU(VAR)3-3 is likely to be a main component in this control because every pole cell nucleus in Su(var)3-3 mutants displays H3K4 methylation and significant reduction of H3K9me2 and H3K9me3. Homozygous Su(var)3-3 mutant females are completely sterile and do not develop oocytes in their egg chambers, which show a spectrum of developmental abnormalities and become arrested already at stage three to four. This suggests that SU(VAR)3-3 is also required during oogenesis in Drosophila. In mammals, two of the three nos homologs are required for female fertility. For nanos-3, a function in primordial germ cell specification was demonstrated in knockout mice. Based on the current data, a likely function for LSD1 during germline specification in mammals is predicted (Rudolph, 2007).

Su(var)3-3 mutant males are sterile and produce immobile sperm, and in primary spermatocytes of Su(var)3-3 males, crystals are frequently found, a phenotype typical for X0 males or XY males carrying a deletion for the crystal (cry) locus. Whether or not SU(VAR)3-3 also interferes with control of activation of Y chromosomal genes in the male germline remains to be studied. Furthermore, alleles of Su(var)3-3 increase crossover in pericentric regions, suggesting that SU(VAR)3-3 is also involved in control of heterochromatin packaging in female meiosis. All these phenotypic effects indicate that the H3K4 demethylase SU(VAR)3-3 is an important regulator in protecting chromatin functions in germline cells (Rudolph, 2007).

Search PubMed for articles about Suppressor of variegation 3-3

Ballas, N., et al. (2001). Regulation of neuronal traits by a novel transcriptional complex, Neuron 31: 353-365. Medline abstract: 11516394

Chen, Y., et al. (2006). Crystal structure of human histone lysine-specific demethylase 1 (LSD1), Proc. Natl. Acad. Sci. 103: 13956-13961. Medline abstract: 16956976

Di Stefano, L., Ji, J. Y., Moon, N. S., Herr, A. and Dyson, N. (2007). Mutation of Drosophila Lsd1 disrupts H3-K4 methylation, resulting in tissue-specific defects during development. Curr. Biol. 17(9): 808-12. Medline abstract: 17462898

Hakimi, M. A. et al. (2002) A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc. Natl. Acad. Sci. 99: 7420-7425. Medline abstract: 12032298

Humphrey, G. W., et al. (2001). Stable histone deacetylase complexes distinguished by the presence of SANT domain proteins CoREST/kiaa0071 and Mta-L1. J. Biol. Chem. 276: 6817-6824. Medline abstract: 11102443

Lee, M. G., Wynder, C., Cooch, N. and Shiekhattar, R. (2005). An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature 437: 432-435. Medline abstract: 16079794

Lee, M. G., et al. (2006). Functional interplay between histone demethylase and deacetylase enzymes, Mol. Cell. Biol. 26: 6395-6402. Medline abstract: 16914725

Metzger, E., et al. (2005). LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437: 436-439. Medline abstract: 16079795

Reuter, G., Dorn, R. and Hoffmann, H.-J. (1982). Butyrate sensitive suppressor of position-effect variegation mutations in Drosophila melanogaster. Mol. Gen. Genet. 188: 480-485. Medline abstract: 6819429

Rudolph, T., et al. (2007). Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol. Cell 26(1): 103-15. Medline abstract: 17434130

Shi, Y., et al. (2003). Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422: 735-738. Medline abstract: 12700765

Shi, Y., et al. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119(7): 941-53. Medline abstract: 15620353

Stavropoulos, G. Blobel and A. Hoelz, Crystal structure and mechanism of human lysine-specific demethylase-1. Nat. Struct. Mol. Biol. 13: 626-632. Medline abstract: 16799558

Szabad, J., Reuter, G. and Schröder, M. B. (1988). The effect of two mutations connected with chromatin functions on female germ-line cells of Drosophila. Mol. Gen. Genet. 211: 56-62. Medline abstract: 3422705

You, A., et al. (2001). CoREST is an integral component of the CoREST- human histone deacetylase complex. Proc. Natl. Acad. Sci. 98: 1454-1458. Medline abstract: 11171972

Wustmann, G., Szidonya, J., Taubert, H. and Reuter, G. (1989). The genetics of position-effect modifying loci in Drosophila melanogaster. Mol. Gen. Genet. 217: 520-527. Medline abstract: 2505058

date revised: 1 November 2007

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