Suppressor of variegation 3-3: Biological Overview | References
Gene name - Suppressor of variegation 3-3
Synonyms - Lsd1, dLsd1, CG17149
Cytological map position-
Symbol - Su(var)3-3
FlyBase ID: FBgn0260397
Genetic map position -
Classification - SWIRM domain, Flavin containing amine oxidoreductase
Cellular location - nuclear
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 (Di Stefano, 2007). 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, dLsd1ΔN was generated (Di Stefano, 2007).
Southern-blot analysis confirmed the authenticity of the dLsd1 alleles. dLsd1ΔN 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 dLsd1ΔN 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 dLsd1ΔN-homozygous flies. Hence dLsd1ΔN 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 dLsd1ΔN-heterozygous animals gave only one-third of the expected number of dLsd1ΔN-homozygous progeny. Interestingly, this reduction in viability is more dramatic in the male progeny (approximately 90% of the viable dLsd1ΔN homozygotes were females) (Di Stefano, 2007).
dLsd1ΔN 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 dLsdΔN 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, dLsd1ΔN 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 dLsd1ΔN and Df(3L)ED4858 recapitulated the phenotypes observed in dLsd1ΔN-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 dLsd1ΔN 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)Stubblevariegated (Sbv), In(1)y3Pyellow, and In(1)whitem4h] 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)Sbv 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)Sbv was crossed to dLsd1ΔN, 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)y3P/+; +; dLsd1ΔN/+ flies showed that a single allele of dLsd1ΔN or dLsd12 suppressed yellow variegation, resulting in a 25% reduction of yellow bristles. Suppression of variegation was also observed with wm4h. 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 dLsd1ΔN 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 dLsd1ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1ΔN 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 dLsd1ΔN 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 dLsd1ΔN 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 dLsd1ΔN 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 dLsd1ΔN mutants. The upregulation occurs late in development, and this may explain the lack of homeotic phenotypes or other early developmental defects in dLsd1ΔN mutants. The stronger changes in histone modification seen in dLsd1ΔN 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 dLsd1ΔN 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).
Su(var)3-3 mutations belong to the strongest dominant suppressors for white gene silencing in the wm4 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 wm4, 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).
Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 [(Suppressor of variegation 3-3), Drosophila ortholog of lysine-specific demethylase 1)] and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. These studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3-9 in promoting heterochromatin spreading at heterochromatin-euchromatin boundaries. Moreover, the data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin (Di Stefano, 2011).
Molecular studies have identified an increasingly large number of histone-modifying enzymes, and biochemical assays readily allow these proteins to be classified, but the more difficult and more important challenge is to understand how these various enzymatic activities are integrated, in vivo, to control biological processes. This study examined the effects of combining mutations in the two H3K4 demethylases Lid and dLsd1 in Drosophila. Thise studies, performed in vivo, show that the interplay between Lid and dLsd1 is dependent on the chromatin context and active signaling pathways. The results show a consistent pattern of genetic interactions between Lid and dLsd1 that is evident in multiple tissues and phenotypes. Unexpectedly, despite their activity as histone H3K4 demethylases, these proteins function antagonistically in a number of functional and developmental contexts. For example, dLsd1 and Lid have opposing functions in the establishment of euchromatin and heterochromatin boundaries. At these locations, the antagonism does not seem to stem from the effects of Lid on H3K4 methylation, but rather from its indirect effects on the spreading of H3K9me2. In addition, while the data show that both Lid and dLsd1 can repress Notch targets within euchromatin when Notch signaling is not active, and that Notch signaling is an important component of the dLsd1 mutant phenotype, genetic evidence supports the hypothesis that Lid and dLsd1 have antagonistic functions in the context of activated Notch signaling. This complex pattern of interactions illustrates that the functional interplay between demethylases, and most likely between other types of chromatin-associated proteins, cannot be rationalized into a single generic model. The evidence that dLsd1 can switch from being a negative regulator of Notch target genes to a positive regulator adds an extra layer of complexity to the interplay between Lid and dLsd1, and strongly supports the concept that the activity of histone demethylases is highly regulated and context-dependent (Di Stefano, 2011).
Genetic and biochemical data support a model for the creation and maintenance of heterochromatin boundaries, proposed by (Rudolph, 2007), in which dLsd1 promotes deacetylation of H3K9 by RPD3 and subsequent methylation of H3K9 by Su(var)3-9, thereby facilitating spreading of heterochromatin. In addition, this study shows an increase in H3K4me1 at the white-rough-est locus in dLsd1 mutant flies, suggesting that active demethylation of H3K4me1 by dLsd1 is an important step in the establishment of heterochromatin. Furthermore, it was found that Lid antagonizes dLsd1 function by promoting euchromatin formation, and that the spreading of heterochromatin seen in Lid mutants is dependent on dLsd1 and Su(var)3-9 activities. Consistent with this notion, H3K9 methylation levels are increased in Lid mutant flies compared with control at the white-rough-est locus and in pericentric heterochromatin. Interestingly, the levels of H3K4me2 and H3K4me3 at the white-rough-est locus are very low and increase only marginally upon Lid mutation, suggesting that Lid function in this context is independent of its histone H3K4 demethylase activity. Previously, Lid had been reported to facilitate activation of Myc target genes in a demethylase-independent manner, and to antagonize Rpd3 histone deacetylase function; moreover, mutation of Lid has been shown to cause a decrease of H3K9 acetylation levels. It is therefore tempting to speculate that Lid opposes the spreading of heterochromatin, independent of its function as a histone H3K4 demethylase, by antagonizing the activity of the dLsd1/Su(var)3-9/Rpd3 complex. This antagonism would explain why, in double mutants for dLsd1 and Lid, the balance between euchromatin and heterochromatin is artificially reset to wild-type levels. Consistently, reorganization of chromatin domains observed in dLsd1 mutant flies affects the expression of genes located at the 2R euchromatin-heterochromatin boundary, an effect that is reversed by mutation of Lid (Di Stefano, 2011).
Given the predominant presence of H3K4 methylation in euchromatin and its important role in determining the transcription status of a gene, it was of interest to establishing the nature of the interplay between Lid and dLsd1 in a euchromatic context. Previous studies had implicated Lid as a crucial factor in the silencing of Notch target genes. The current study shows a cooperative role for Lid and dLsd1 in repressing Notch target gene expression, and suggests that they contribute to repression by maintaining low levels of H3K4 methylation. Repression of Notch target genes is essential for the establishment of Notch-inhibited cell fates, suggesting that Lid and dLsd1 could play a role in proper cell fate specification during Drosophila development. Interestingly, the role of dLsd1 does not seem to be limited to repression of Notch target genes. Indeed, genetic analysis suggests that, in a context in which the Notch signaling pathway is active, dLsd1 switches from a repressor to an activator role. Such a dual role had already been described for Su(H), whose switch from a repressor to an activator has been suggested to be mediated through an exchange of associated proteins. Similarly, in mammalian cells, studies have shown that LSD1 activity can be modulated by changes in composition of the complexes present at the Gh promoter, and, depending on the cell type (somatotroph or lactotroph), LSD1 can act as either an activator or a repressor. Therefore, a possible explanation for the current data is that, depending on the complexes available, dLsd1 can switch from being a repressor to acting as an activator of Notch target genes. Alternatively, dLsd1 mutation could promote derepression of negative regulators of Notch activity, or could directly modulate Notch activity by demethylating crucial components of the Notch-activating complex. Further studies are required to distinguish between these possibilities (Di Stefano, 2011).
These results provide the basis for future studies aimed at investigating whether the dual role of dLsd1 in modulating Notch signaling is conserved in mammals. In mice, LSD1 has been shown to repress the Notch target Hey1 in late stages of pituitary development, suggesting that its ability to regulate Notch target genes is conserved. This pathway-specific function of LSD1 could potentially be exploited to create novel strategies to manipulate Notch-mediated carcinogenesis (Di Stefano, 2011).
Collectively, these results reveal an intricate interplay between the histone demethylases Lid and dLsd1 in the control of higher-order chromatin structure at euchromatin and heterochromatin boundaries affecting developmental gene silencing. They also demonstrate an involvement of dLsd1 and Lid in Notch pathway-specific control of gene expression in euchromatin, and support the idea that, depending on the context, Lid and dLsd1 can favor either transcriptional activation or transcriptional repression (Di Stefano, 2011).
The conserved SWI/SNF chromatin remodeling complex uses the energy from ATP hydrolysis to alter local chromatin environments through disrupting DNA-histone contacts. These alterations influence transcription activation, as well as repression. The Drosophila SWI/SNF counterpart, known as the Brahma or Brm complex, has been shown to have an essential role in regulating the proper expression of many developmentally important genes, including those required for eye and wing tissue morphogenesis. A temperature sensitive mutation in one of the core complex subunits, SNR1 (SNF5/INI1/SMARCB1), results in reproducible wing patterning phenotypes that can be dominantly enhanced and suppressed by extragenic mutations. SNR1 functions as a regulatory subunit to modulate chromatin remodeling activities of the Brahma complex on target genes, including both activation and repression. To help identify gene targets and cofactors of the Brahma complex, advantage was taken of the weak dominant nature of the snr1E1 mutation to carry out an unbiased genetic modifier screen. Using a set of overlapping chromosomal deficiencies that removed the majority of the Drosophila genome, genes were sought that when heterozygous would function to either enhance or suppress the snr1E1 wing pattern phenotype. Among potential targets of the Brahma complex, components were identified of the Notch, EGFR and DPP signaling pathways important for wing development. Mutations in genes encoding histone demethylase enzymes were identified as cofactors of Brahma complex function. In addition, it was found that the Lysine Specific Demethylase 1 gene (lsd1) was important for the proper cell type-specific development of wing patterning (Curtis, 2011).
Although chromatin remodeling is an important component of gene activation, its role in gene repression is not as well understood. The unbiased genetic screen using a weak dominant temperature sensitive mutant allele of a key Brm complex regulatory subunit has provided new insights into the involvement of chromatin remodeling complexes in developmental tissue patterning. Mutations in components of several signaling pathways, including Notch, EGFR and DPP/TGFβ, genetically interacted in these assay. These results, combined with candidate gene genetic analyses, have confirmed a previous hypotheses that the Brm complex participates in both gene activation and gene repression to help coordinate several key signaling pathways that lead to proper animal patterning. The results are largely concordant with the results of previous limited screens that identified a set of dominant modifiers of brmK804R mutant phenotypes. Among 14 chromosomal deficiencies that enhanced the brmK804R rough eye phenotype, this study found that 6/14 were also dominant enhancers of the snr1E1 wing phenotype and 3/14 were suppressors, suggesting that dominant modifier screens are effective tools for identifying unknown loci important for Brm complex regulatory functions. Consistent with this view, the Brm complex has been shown to interact the Notch ligand, Delta, in the developing fly eye. The genetic modifier screen results presented in this study indicate that Notch signaling functions may also be mediated through the Brm complex in the developing fly wing. Given the strong evolutionary conservation of these pathways, it is anticipated that the vertebrate SWI/SNF orthologs will play a similarly important role in patterning the tissues of vertebrate animals (Curtis, 2011).
What are the target genes regulated by the Brm complex in the developing wing? Previous studies have found that loss of snr1 function results in ectopic dpp and rhomboid expression in intervein cells. These data are consistent with the genetic interactions shown in this report that were observed using mutants affecting both the DPP and EGFR pathways. These studies have additionally provided an important insight into gene regulatory factors beyond signaling pathways that contribute to transcription repression in collaboration with chromatin remodeling complexes at key points in the development and differentiation of tissues. In the present analyses, several lines of evidence are provided suggesting that the mechanism of Brm complex-mediated gene repression is not only dependent upon a tight, physical and genetic relationship between two core subunits, SNR1 and MOR, but also on histone lysine demethylase enzymes (Curtis, 2011).
It has been reported that the full in vitro chromatin remodeling activity of the mammalian BRM/BRG1 complex on reconstituted nucleosomes can be accomplished with a subset of three or four core components, including the SNF5 (SNR1), BAF155/BAF170 (MOR) and BRM/BRG1 ATPase subunits that are highly conserved from yeast to vertebrates. Each of these subunits is required for complex stability in vivo as RNAi depletion of the individual components in cultured Drosophila cells leads to reduced stability of the other subunits with corresponding changes in target gene expression. Loss of BRM function in vivo, using either a dominant negative ATPase deficient mutant (brmK804R) or an amorphic allele (brm2), can suppress the snr1E1 wing phenotype revealing an important role for SNR1 in restraining Brm complex transcription activation functions. In contrast, mor mutants enhance mutant phenotypes associated with reduced brm function and show allele-specific interaction with snr1E1, suggesting an important functional relationship between the MOR, BRM and SNR1 subunits. MOR likely serves as a scaffolding protein, since physical associations were observed between SNR1-MOR and MOR-BRM. Two independent domains of MOR, the SWIRM and SANT, domains respectively, are critical for the binding interaction. Therefore, the contribution of SNR1 regulatory function on Brm complex chromatin remodeling activities may depend on crosstalk through MOR since no direct physical contacts between SNR1 and the BRM subunit have been observed (Curtis, 2011).
An unbiased dominant modifier genetic screen allowed identification of histone lysine demethylase enzymes as novel coregulators of the Brm complex in controlling gene expression. Previous screens looking for modifiers of a brm dominant negative allele (brmK804R) did not uncover mutations in histone-modifying families, such as acetyltransferases, deacetylases, and methyltransferases. However, the wing patterning defect associated with snr1E1 is highly sensitive, allowing observation of subtle changes in remodeling activities, and identification a family of epigenetic modifiers as potential Brm regulators. Previous studies have found that histone deacetylases (HDACs) were important corepressors that worked in direct collaboration with the Brm complex. In the present study, mutations in predicted demethylase genes genetically interacted with snr1E1 and LSD1 was shown to associate with the Brm complex in vivo, suggesting demethylases are also potential cofactors. While a functional cooperation between histone deacetylation and demethylation activities has been suggested previously, the current data implicates at least three chromatin modifying activities—ATP-dependent chromatin remodeling, histone deacetylation and demethylation—cooperating to regulate tissue-specific gene repression through multiple bridging interactions. In this scenario, the commitment of a gene promoter to be repressed in a cell type-specific manner would depend on the collateral influence of several chromatin modifying activities that would serve to help establish a repressed transcriptional environment, refractory to the influence of signaling pathways operational in adjacent cells (Curtis, 2011).
There appears to be no correlation between the predicted demethylase lysine substrate and enhancement/suppression of the snr1E1 phenotype. This is not surprising, since a high degree of functional redundancy exists amongst demethylase enzymes. It is likely that multiple demethylase enzymes cooperate to regulate a variety of target genes. This is supported by experimental evidence showing that knockdown experiments of individual demethylases, for example lsd1, in cell culture often showed little or no change in global methylation status, though significant changes were observed on a gene-specific level in vivo. Independent loss of function mutations in two JARID family members, lid and Jarid2/CG3654, resulted in an opposite genetic interaction with snr1E1. This study observed that a loss of function mutation in lid, (lid2) dominantly suppressed, whereas a loss of function mutation in Jarid2 (CG3654EY02717) enhanced the ectopic vein phenotype associated with snr1E1. LID is an H3K4me3/me2 specific demethylase. JARID2 is predicted to have the same substrate specificity, though overexpression analyses in cell culture experiments showed no global increase in H3K4me3/2. The observed opposite genetic interaction with snr1E1 may reflect differences in target gene regulation by LID and JARID2, either as a consequence of different target genes controlled in the developing wing or through opposite mechanisms in controlling gene transcription. Importantly, JARID2 homologs in Xenopus and mammalian model systems physically associate with the Polycomb Repressor Complex-2 (PRC2) and directly contribute to transcriptional repression by preventing the methylation of the histone lysine residues correlated with transcriptional activation. Therefore, mutation of JARID2 (CG3654EY02717) may enhance the snr1E1 phenotype if the normal role of CG3654 is to suppress transcription of a particular gene involved in wing vein development (Curtis, 2011).
The cell-fate decision to become vein or intervein is largely based on cell-type specific expression of transcription factors. In vein cells, transcription factors with gene targets that promote vein development are highly expressed, whereas those with gene targets that block vein fate are repressed. In intervein cells, the opposite is observed, with heightened expression intervein-promoting factors and decreased expression of vein promoting factors. The Brm complex has an important role in development of both cell fates, serving a positive role to promote vein development in vein cells, and repress vein development in intervein cells. The opposite genetic interaction phenotypes observed with lid and Jarid2 could be partially explained if the Brm complex is coordinating with the each specific demethylase to regulate different target genes. This study found that loss of function mutations in vein promoting genes, such as Egfr, suppressed the snr1E1 phenotype. The results suggest that LID and EGFR may regulate the expression of similar target genes and indeed EGFR (as well as other signaling pathways) may function in wing vein development through LID. In this scenario, a loss of function mutation in lid would result in a decrease in the expression of vein promoting genes, thereby suppressing the snr1E1 ectopic vein phenotype. Enhancement of the snr1E1 phenotype by Jarid2/CG3654EY02717 can be explained if JARID2 promotes activation of genes required to block vein differentiation, just as loss of function mutations in vein-inhibiting factors, such as net, enhanced the snr1E1 phenotype (Curtis, 2011).
The candidate genetic screen results suggest that histone lysine demethylase enzymes are likely cofactors of Brm chromatin remodeling activity. However, it is highly unlikely that stable physical associations are made between the complex and all six demethylases. The possibility cannot be eliminated that the Brm complex and demethylase enzymes are independently regulating genes involved in wing patterning or eliciting their functions on different targets at different times during development to contribute to the final read-out of vein/intervein patterning in the adult wing. However, a direct physical association was detected between the Brm complex and LSD1 in coimmunoprecipitation and GST-pulldown experiments, implying that LSD1 is a potential cofactor of Brm complex remodeling activities (Curtis, 2011).
The genetic epistasis experiments demonstrated an important in vivo functional relationship between LSD1 and the core subunits of the Brm complex, SNR1, MOR, and BRM. Brm complexes can be subdivided into two groups: PBAP complexes contain BAP170, POLYBROMO/BAP180, and SAYP, whereas BAP complexes contain OSA. These complexes can regulate target genes in a synergistic, antagonistic, or independent manner. BAP and PBAP complexes likely have differential regulatory functions, since they have distinct, but overlapping, localization patterns on larval salivary gland polytene chromosomes and targeted knockdown of OSA, POLYBROMO, or BAP180 using RNAi in cultured Schneider cells, leads to differential expression profiles on whole genome arrays. OSA, BAP170, BAP180, and SAYP likely have different roles in development, as mutation of each leads to different abnormalities. For example, BAP180 is required for proper egg shell development, whereas BAP170 is necessary to stabilize BAP180, important for adult viability, and vein cell differentiation. OSA is necessary for photoreceptor development, normal embryonic segmentation, and wing patterning. BAP, but not PBAP complexes have an important role in regulating cell cycle progression through mitosis (Curtis, 2011).
In mice, knockout of Baf180 causes misregulation of retinoic acid receptor target genes and heart developmental defects, indicating that PBAP complexes may have a role in nuclear receptor transcriptional regulation. The LSD1 corepressor complex, including the cofactor proteins, CoREST, and histone deacetylase, HDAC1/2, have also been indicated in nuclear receptor transcriptional regulation. LSD1 association in complexes containing the Estrogen Receptor (ER) or Androgen Receptor (AR) leads to a switch in methylated lysine specificity, and results in demethylation of mono- and dimethylated H3K9 and gene activation (Curtis, 2011).
It is not known how BAP vs. PBAP complexes are differentially recruited to target genes. Recruitment of BAP complexes to specific target genes may depend on the physical associations made by OSA and sequence-specific transcription factors. For example, OSA is required for expression of target genes associated with the transcription factors Pannier and Apterous and can promote transcriptional repression of genes regulated by Wnt/Wingless signaling. Genetic epistasis experiments reveal that LSD1 cooperates with PBAP, but not BAP containing complexes in the Drosophila wing, suggesting that the physical association observed between LSD1 and Brm complex may be limited to PBAP complexes and provide a mechanism for selective target gene recruitment and regulation by Brm remodeling complexes. Further analyses, such as GST-pulldown and coimmunoprecipitation experiments using PBAP specific components need to be performed to address this possibility (Curtis, 2011).
Ectopic vein development within intervein tissue can result from two different possibilities: 1) the loss of a factor necessary to block vein cell development, or 2) the gain of a factor that promotes vein cell differentiation. Knockdown experiments suggest LSD1/dCoREST functions through the first mechanism. Loss of LSD1/dCoREST throughout the entire developing wing imaginal disc resulted in the development of vein material in intervein tissue, but no changes in vein morphology were observed. If LSD1/dCoREST normally functioned to promote vein development, then loss throughout the entire wing should have led to a loss of vein phenotype (Curtis, 2011).
Several lines of evidence suggest that LSD1 may be capable of regulating gene transcription in a cell-type or stage dependent manner. The affect of homozygous loss of lsd1 on transcriptional regulation of known target genes, including the Sodium Channel and NicotinicAcetylcholine Receptor-β is minimal in embryos and larvae, but significant in pupae. This implies that LSD1 has an important role in regulating gene transcription during later developmental stages. Moreover, LSD1 negative regulation of the homeobox genes, Ultrabithorax (Ubx) and abdominal-B (abd-B) continues into adulthood, as lsd1 null animals display significantly increased expression of these genes as the animals continue to age. This stage-dependent requirement appears to be conserved, as the conditional knock-out of LSD1 in the developing mouse pituitary gland causes little or no morphological defects early in pituitary development (E9-9.5), but significantly alters cell-fate determination choices during later stages (E17.5). Furthermore, LSD1 mediates both gene activation and gene repression of different target genes by associating with several multisubunit complexe (Curtis, 2011).
Knockdown and genetic epistasis experiments further support the idea that LSD1 is important for regulating terminal differentiation, since patterning phenotypes are similar to those observed with defects in DPP and EGFR signaling, the pathways active during pupal development, rather than observed with defects in HH signaling, an early pathway component. Previous work has demonstrated an important role in Brm complex involvement in EGFR, DPP, and Delta/N signaling. More recently, it has been demonstrated that OSA, the defining subunit of the BAP complex, is required to activate EGFR targets in the developing wing. In this regard, the Brm complex may be cooperating with LSD1 to regulate several conserved signaling pathways, but this cooperation may be tissue and developmental time-point dependent (Curtis, 2011).
The choice and timing of specific developmental pathways in organogenesis are determined by tissue-specific temporal and spatial cues that are acted upon to impart unique cellular and compartmental identities. A consequence of cellular signaling is the rapid transcriptional reprogramming of a wide variety of target genes. To overcome intrinsic epigenetic chromatin barriers to transcription modulation, histone modifying and remodeling complexes are employed. The deposition or erasure of specific covalent histone modifications, including acetylation, methylation, and ubiquitination are essential features of gene activation and repression. This study has found that the activity of a specific class of histone demethylation enzymes is required for the specification of vein cell fates during Drosophila wing development. Genetic tests revealed that the Drosophila LSD1-CoREST complex is required for proper cell specification through regulation of the DPP/TGFβ pathway. An important finding from this analysis is that LSD1-CoREST functions through control of rhomboid expression in an EGFR-independent pathway (Curtis, 2012).
The Su(var)3-3 gene (CG17149) encodes the Drosophila LSD1 homolog. Mutations in Su(var)3-3 result in aberrant histone methylation and heterochromatin formation, with increased global levels of H3K4me2 and impaired heterochromatic gene silencing. A physical association between LSD1 and CoREST has been described in Drosophila (Dallman, 2004), revealing that the critical relationship between these proteins is conserved. LSD1 has an important role in organogenesis and germ line maintenance, such as during mouse anterior pituitary development (Wang, 2007) and Drosophila ovary and wing development. LSD1 also regulates neural stem cell proliferation by modulating signaling via the orphan nuclear receptor TLX (Sun, 2010)¸ and LSD1 appears to have distinct functions in mammalian neuronal morphogenesis (Fuentes, 2012; Zibetti, 2010) as well as stem cell self-renewal and differentiation (Adamo, 2011). In humans, loss of LSD1 has been strongly correlated with several types of cancer and high-risk tumors, including prostate cancer, breast cancer and neuroblastomas). In contrast, overexpression of LSD1 has also been linked to some cancers. As a consequence of the emerging links between histone demethylase functions and disease, an understanding how LSD1 contributes to specific cell-cycle regulation and developmental processes is crucial (Curtis, 2012).
The Drosophila wing provides an outstanding in vivo model system to identify factors that regulate cell-fate determination as alterations in cell-fate can often be observed at the single cell level. Multiple conserved signaling pathways contribute to wing patterning and development and are regulated, in part, by the coordinated activities of chromatin remodeling complexes and epigenetic modifying enzymes. Previously work has identified histone lysine demethylase enzymes as coregulators of Brm complex remodeling activities in a genetic screen for factors that influenced a wing patterning phenotype associated with a conditional loss-of-function mutation in the snr1 gene that encodes a core regulatory subunit of the Brm complex. Genetic interaction tests indicated that lsd1 (Su[var]3-3) most likely interacted with the PBAP subtype of the Brm complex (Curtis, 2011). This report further addresses how LSD1 contributes to the cell-type and developmental time-point specific regulation of conserved signaling pathways by understanding its contribution to wing patterning and development (Curtis, 2012).
Recently, it was suggested that LSD1 regulates notch signaling during Drosophila wing development (Mulligan, 2011). This study presents evidence from genetic interaction analyses and tissue or cell-type specific targeted depletion experiments that suggest LSD1 and CoREST/CG42687 (synonymous with CG33525) may also regulate the DPP/TGFβ signaling pathway in a noncanonical manner, by regulating expression of rhomboid, a key player in canonical EGFR signal transduction. This is the first demonstration of LSD1-CoREST regulated DPP/TGFβ signaling and the results further define important roles of the LSD1-CoREST complex in tissue patterning (Curtis, 2012).
The appropriate elaboration of wing vein and intervein cell fates depends on the interplay of factors that promote and those that repress or block vein cell differentiation. In this study, we provide genetic evidence suggesting an important role for lsd1 and CoRest in repressing vein-promoting genes in intervein cells. Ectopic vein development can result from either the loss of a factor required for repressing vein cell differentiation or the gain of a factor that promotes vein cell fate in intervein cells. The experimental results suggest that lsd1 and CoRest utilize the first mechanism, since the aos hypomorphic mutation (aosw11), a factor known to repress vein fate, is enhanced by CoRestEY14216 and lsd1ΔN and targeted depletion by shRNAi of lsd1 and CoRest throughout the entire developing wing imaginal disc resulted in ectopic veins rather than loss of vein phenotypes. It was reasoned that if the LSD1-CoREST complex normally functions as a positive factor to promote vein development as proposed by the second mechanism, then mutations in lsd1 and CoRest or shRNAi depletion in the wing imaginal disc should produce a loss of vein phenotype. Based on the evidence presented in this manuscript, and on the recent finding that LSD1 is important for the regulation of NOTCH signaling in the wing (Di Stefano, 2011), it is proposed that the requirements of LSD1-CoREST are temporal and cell-type specific, and possibly dependent on the physical associations between LSD1 and several multiprotein complexes (Curtis, 2012).
An elaborate signaling network regulates wing patterning, where considerable cross-talk and functional redundancy connects five developmental pathways. For example, during pupal development, the main role of EGFR and DPP activation is to coordinately promote and maintain differentiation into vein cells while NOTCH activation establishes the provein-intervein boundary. However, DPP and NOTCH pathways are codependent, since expression of the NOTCH ligand, DELTA (DL) and its downstream target, ENHANCER OF SPLIT, (E(spl)mβ), require DPP signaling. LSD1 has been shown to interact directly with the histone deacetylase SIRT1 to repress NOTCH targets, suggesting important epigenetic functions for these co-repressors in metazoan development. However, recently it was shown that CoREST could function as a positive regulator of NOTCH in Drosophila follicle cells and wings (Domanitskaya, 2012). Therefore, there is growing precedent for the LSD1-CoREST complex to have both positive and negative roles in regulating gene expression depending on developmental context (Curtis, 2012).
LSD1 and CoREST depletion in the developing wing causes bifurcated or duplicated crossveins, a phenotype previously observed with Hairless (H) loss of function mutations. Because H both antagonizes NOTCH and promotes EGFR signaling, it is difficult to decipher the individual pathway regulated by LSD1-CoREST. Furthermore, the broadened vein delta phenotype observed at the wing margin in wing-specific LSD1-CoREST depleted and lsd1ΔN null flies (Di Stefano, 2011) is similar to Notch and DPP receptor (tkv) loss of function phenotypes (Curtis, 2012).
It is proposed that during the initial stages of wing vein development and differentiation, LSD1 negatively regulates NOTCH signaling. This is based on the observation that loss of lsd1 function suppresses the notched wing phenotype associated with mutations in suppressor of hairless (Su[HT4]) (Mulligan, 2011). However, later in development during vein refinement and maintenance, LSD1 appears to undergo a regulatory switch to positively regulate NOTCH signaling, since lsd1ΔN suppresses the short vein phenotype associated with the gain-of-function NAx-16 mutation. Additionally, the increased expression of downstream E(spl) targets in NAx-16 mutants is reversed by lsd1ΔN (Di Stefano, 2011). It was also recently shown that a transheterozygous mutant allele of CoRest (CoRestGF60) could enhance the wing phenotypes of flies carrying alleles of Dl and N (Domanitskaya, 2012), suggesting positive functions in regulating NOTCH signaling. Concurrently, LSD1 and CoREST repress vein cell differentiation by regulating components of the DPP signaling pathway at multiple points. For example, lsd1ΔN and CoRestEY14216 genetically interact with both dpp and genes encoding its receptors (e.g., dpp, tkv, sax), consistent with upstream functions. Strong genetic interactions were observed with downstream DPP signaling components (e.g., mad, med, ara, caup, shn), which suggests that the LSD1-CoREST complex has important regulatory functions in controlling the expression of DPP pathway targets. This conclusion is further supported by ectopic expression of the DPP-specific downstream signaling component, p-MAD, was observed in LSD1-CoREST-depleted animals. Activated DPP signaling is confined to proveins largely by the overexpression of TKV, a member of the TGFβ receptor family, in intervein boundary cells. TKV binds and sequesters the DPP morphogen. When TKV is downregulated, DPP spreads into regions of the wing destined to become intervein cells, resulting in ectopic veins. It is predicted that TKV is the most likely target of LSD1-CoREST complex regulation, since genetic interactions were observed between lsd1ΔN and CoRestEY14216 and almost all loss of function mutations in DPP signaling components, and tissue-specific LSD1-CoREST depletion lead to the development of ectopic veins, similar to phenotypes observed with loss of function alleles of tkv. Because activation of NOTCH and repression of DPP signaling are both required to repress vein promoting genes in differentiating intervein cells, LSD1 appears to have cell-type and context-specific activities to differentially regulate these pathways (Curtis, 2012).
Coimmunoprecipitation experiments suggested a complex forms between the HDAC1/2 class protein RPD3, LSD1, CoREST, and two TTK splice variants TTK88 or TTK69 (Dallman, 2004). Complexes containing CoREST/TTK69 or CoREST/TTK88 independently localize on polytene salivary glands, suggesting differential gene targeting (Dallman, 2004). TTK and REST are likely functional homologs. Orthologs of tramtrack only exist in invertebrates, whereas REST orthologs are vertebrate-specific (Dallman, 2004). TTK69 is a transcription factor that can recognize and bind to a specific DNA RE-1 consensus sequence (CCAGGACG), resulting in gene transcription (Dallman, 2004). Unpublished observations suggest that TTK69, but not TTK88, function to negatively regulate vein cell development, since an incomplete vein phenotype is observed when TTK69 is overexpressed, whereas overexpression of TTK88 results in the development of ectopic veins. Therefore, it is predicted that LSD1-CoREST-TTK69 form a complex in developing wing tissue to negatively regulate DPP signaling in intervein cells. Furthermore, in mammals, the Brg1 complex chromatin remodeling capacity and recruitment specificity depends on formation of a LSD1-CoREST-REST-BRG1 complex (Ooi 2006). Because LSD1 can physically associate with the Brm chromatin remodeling complex in Drosophila (Curtis, 2011), it is predicted that the Brm complex-LSD1-CoREST-TTK69 super-complex regulates genes essential for wing patterning, possibly through co-localization or recruitment to RE-1 consensus binding sites. Intriguingly, RE-1 consensus sites are present in both the rho and tkv gene loci, making these exciting targets for future investigation (Curtis, 2012).
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. This study examined the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, dhtt was found to act as a suppressor of position effect variegation (PEV), suggesting that it influences chromatin organization. dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, a candidate genetic screen was constructed using RNAi lines targeting known PEV modifier genes. dhtt was found to modify phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the histone demethylase dLsd1, in both the ovary and wing, and dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla (Dietz, 2015).
Previous studies of the inverse relationship between the age at onset of clinical symptoms and the size of the HTT CAG repeat mutation have revealed that the repeat confers on the mutant allele a fully dominant gain-of-function property. However, it is unknown whether this is the acquisition of enhanced normal huntingtin function or the acquisition of a novel opportunistic function, although targeted null and CAG expansion mutations at the mouse homolog provide support for both possibilities. Despite earlier studies, details of the normal function of huntingtin remain relatively elusive, hindering further investigation into the molecular mechanisms underlying the disease (Dietz, 2015).
This study uses a novel dhtt allele to examine the normal function of Drosophila huntingtin, focusing on its potential role in chromatin function during development. Although dhtt flies are viable and appear grossly normal, genetic findings indicate that dhtt influences chromatin regulation: (i) dhtt acts as a suppressor of PEV, suggesting that it is involved in heterochromatin formation; (ii) dhtt affects heterochromatin spreading in a PEV model; (iii) dhtt genetically interacts with a number of genes encoding proteins known to affect chromatin organization and function and (iv) dhtt genetically interacts with the HDM dLsd1 and facilitates its ability in demethylating histone H3K4 (Dietz, 2015).
PEV is a powerful genetic assay that has been used previously to identify genes that can regulate chromatin structure. In PEV models, a chromosomal rearrangement or transposition abnormally juxtaposes a reporter gene with heterochromatin. A variegated phenotype is produced since the gene is stochastically silenced in some of the cells in which it is normally active. The silencing that occurs in PEV is attributed to the ‘spreading’ of heterochromatin along the chromosome into a region that would normally be in a euchromatic form. Thus, since the reporter gene is on the boundary between these two states, PEV provides a sensitive system in which to test genetic modifiers of heterochromatin formation. This study uses two independent PEV assays [T(2;3)Sbv and In(1)y3P] and demonstrates that dhtt facilitates heterochromatin formation, thereby suppressing variegated phenotypes. To date, approximately 500 dominant Su(var) and E(var) mutations have been isolated from PEV screens and it is estimated that these affect about 150 unique genes. Those that have been molecularly characterized so far have been revealed to generally encode chromosomal proteins or modifiers of chromosomal proteins (Dietz, 2015).
Histone post-translational modifications (PTMs) play essential roles in the transition between active (euchromatin) and inactive (heterochromatin) chromatin states. In particular, histone methylation has been widely studied in nearly all model systems and is generally recognized as an epigenetic marker for transcriptionally silent heterochromatin. High levels of methylated histone H3K9me2 are associated with heterochromatin loci. Using the established PEV model, wm4, this study demonstrates that the dhtt allele dominantly reduces the level of histone H3K9me2 at the white locus and the adjacent CG12498 gene at the heterochromatin–euchromatin boundary. This level of histone H3K9me2 reduction is comparable to that caused by a dLsd1/Su(var)3-3 null allele, an established suppressor of variegation. The loss of dhtt therefore significantly influences chromatin structure, thereby shifting the euchromatin–heterochromatin boundary (Dietz, 2015).
Where in the cell might huntingtin function to affect chromatin structure and act as a suppressor of variegation? Although the majority of huntingtin in human and mouse cells has been shown to reside within the cytoplasm, about 5% is estimated to be nuclear. A previous report suggests that Drosophila huntingtin is solely cytoplasmic, but this was based solely on ectopic dhtt overexpression. Since both fly and mouse loss-of-function huntingtin models show defects in mitotic spindle orientation in neuroblast precursors, it is clear that huntingtin does have a nuclear function. However, it is possible that dhtt could also influence chromatin structure by acting in the cytoplasm (Dietz, 2015).
Based on the findings that dhtt dominantly suppresses PEV and affects chromatin function, it was hypothesized that it may genetically interact with previously identified PEV modifiers. The approach to screening for possible dhtt interactors utilized a collection of RNAi lines targeting known suppressors and enhancers of PEV. Such a screen has a number of caveats: first, it relies on RNAi producing a modifiable phenotype in a relevant tissue. Secondly, due to the nature of the screen, it is largely limited to looking for interactions in the adult eye and wing. Interestingly, in some cases, interactions between dhtt and genes in the wing were found, but not in eye and vice versa. This may reflect tissue-specific requirements for different genes, or that dhtt functions only within certain complexes in certain tissues. Nevertheless, a number of strong genetic interactors of dhtt were identified, which included central regulators of chromatin architecture and function, such as the heterochromatin proteins, HP1a and HP1b, brm—the ATPase subunit of the SWI/SNF (Brm) complex, the transcription factor dE2F1 and various HDMs and HMTs. Next, the interaction between dhtt and dLsd1 was evaluated for the following reasons: dLsd1 interacts with both the dhtt allele and dhtt RNAi and loss of dhtt causes enhancement of dLsd1 phenotypes in both wing and ovary. Additionally, it was found that dhtt and dLsd1 both affect PEV and heterochromatin formation to comparable extents (Dietz, 2015).
Although dhtt-deficient flies are fertile and display no obvious ovarian phenotype, loss of dhtt strongly enhances the dLsd1 ovary defects. It was hypothesized that dLsd1 and dhtt collaborat in the regulation of histone H3K4 methylation at specific loci to control gene expression critical for oogenesis. Similarly, in contrast to dLsd1 mutant flies which show elevated levels of histone H3K4me1 and H3K4me2, any changes in the global levels of these modifications in dhtt-deficient flies could not be detected. However, simultaneous knockdown of dLsd1 and dhtt results in a significant increase in histone H3K4me1 and H3K4me2 levels over that of the dLsd1 knockdown alone. Human LSD1 is a component of the CoREST/REST (repressor element silencing transcription factor) complex, which represses the transcription of neuronal genes in non-neuronal cell lineages. Within this complex, LSD1 acts to demethylate histone H3K4 residues in nucleosomes at REST target genes, thereby contributing to their transcriptional repression. Mammalian full-length huntingtin has been shown to physically interact with this complex and contribute to its regulation, and it will therefore be interesting to determine whether dLsd1 and dhtt similarly associate with each other. Unfortunately due to the lack of phenotype upon knocking down the Drosophila ortholog of CoREST, dCoREST, with the available RNAi lines, testing for a potential interaction with dhtt could not be performed by this study. The genetic interaction between dhtt and dLsd1 could potentially account for the strong effect of dhtt seen on H3K9 methylation at the heterochromatin/euchromatin boundary in the wm4 PEV model. dLsd1 has been shown to physically associate with Su(var)3-9 and to control Su(var)3-9-dependent spreading of histone H3K9 methylation along euchromatin (Dietz, 2015).
There is considerable evidence suggesting a link between aberrant acetylation and methylation marks and HD. Mouse Htt has been implicated in facilitating the trimethylation of histone H3K27 in developing murine embryoid bodies. The levels of histone H3K4me3 have been shown to change at dysregulated promoters in a mouse HD model (R6/2) and human HD postmortem brain tissue. The screen in this study uncovered interactions between dhtt and Drosophila HDM and HMTs with a variety of different histone H3 specificities (H3K4, H3K27, H3K9 and H3K79). It is therefore possible that dhtt has a general role, possibly as a scaffold protein, in facilitating a number of complexes containing histone-modifying enzymes with different specificities. Since mammalian full-length huntingtin has been implicated in the trimethylation of histone H3K27 by facilitating PRC2 function, it is surprising that the histone H3K27 methyltransferase, esc, is the only component of PRC2 found to interact with dhtt. Although there was no effect of the dhtt null mutation on the global levels of histone H3K27me levels, it is possible that dhtt may play a similar role to mouse Htt in modulating histone H3K27me during development, with histone H3K27me differences only observed at specific loci (Dietz, 2015).
A number of the dhtt interacting genes found in the screen encode for important chromatin regulating proteins that have previously been found to genetically interact with each other. For example, a strong interaction between dhtt and brm, the central subunit of the Brm SWI/SNF complex, was detected. brm is known to interact with E(Pc), dE2F1, Asx and Rpd3, which were also found in the screen. The SIN3 corepressor complex is a class I HDAC complex conserved from Drosophila to humans and regulates gene transcription through deacetylation of nucleosomes. Loss of dhtt suppresses both eye and wing phenotypes caused by Sin3A RNAi. Drosophila Sin3A has been shown to interact with the HDAC Rpd3 and the HDM, lid—both of which were also scored as hits in the screen. Furthermore, mammalian Sin3A was previously reported as a huntingtin N-terminal yeast two-hybrid interactor (Dietz, 2015).
Drosophila has proved to be a useful model to investigate polyglutamine-fragment toxicity. Expression of an N-terminal fragment with an expanded polyglutamine tract in the fly has been shown to accumulate in the nucleus. It would therefore be interesting to evaluate whether the normal chromatin regulatory functions of dhtt are perturbed in the fly polyglutamine-fragment models. Although the Drosophila screen in this study was designed to look initially for phenotypes in visible external structures of the adult fly (wing and eye), many of the genes that were found to interact with dhtt are known to also be expressed and function in the developing nervous system. It is therefore possible that dhtt may also exert a role in regulating chromatin function during neurogenesis and neural function in the fly, leading to subtle behavioral mutant phenotypes that have been described previously. This study establishes Drosophila as a system in which to investigate the normal role of dhtt in chromatin regulation. It will be particularly useful in examining dhtt functions that are evolutionarily conserved as these provide assays with which to determine the impact of the expanded polyglutamine region on full-length huntingtin function, thereby deepening our understanding of the mechanism that initiates the HD disease process (Dietz, 2015).
The Lysine (K)-specific demethylase (LSD1) family of histone demethylases regulates chromatin structure and the transcriptional potential of genes. LSD1 is frequently deregulated in tumors and depletion of LSD1 family members causes developmental defects. This study reports that reductions in the expression of the Pumilio (PUM) translational repressor complex enhances phenotypes due to dLsd1 depletion in Drosophila. The PUM complex is a target of LSD1 regulation in fly and mammalian cells and its expression is inversely correlated with LSD1 levels in human bladder carcinoma. Unexpectedly, PUM was found to post-transcriptionally regulate LSD1 family protein levels in flies and human cells indicating the existence of feedback loops between the LSD1 family and the PUM complex. These results highlight a new post-transcriptional mechanism regulating LSD1 activity and suggest that the feedback loop between LSD1 family and the PUM complex may be functionally important during development and in human malignancies (Miles, 2015).
This study has identified a novel regulatory mechanism between the KDM1 family of histone demethylases and the PUM post-transcriptional repressor complex. Specifically, it was found that the components of the PUM complex are directly bound by LSD1 in flies, mice and humans. In addition, a conserved regulatory feedback loop was discoved between the PUM complex and members of the LSD1 family. It is proposed that LSD1 regulates the expression of the PUM complex and that PUM post-transcriptionally fine-tunes the translation of dLsd1 and LSD2. Importantly, these studies suggest that this interplay is physiologically relevant as Pumilio and dLsd1 have synergistic roles during Drosophila development (Miles, 2015).
In support of this hypothesis, it was found that the concomitant depletion of dLsd1 and components of the Pumilio complex in Drosophila results in synthetic lethality. In addition, a strong enhancement of the dLsd1-RNAi wing phenotype was found when components of the Pumilio complex specifically were co-depleted in the wings. These findings suggest that dLsd1 and Pum act synergistically to regulate cell fate and cell survival decisions during Drosophila development and are in agreement with previous findings in C. elegans. The site-specific effect on wing formation might be dependent on gradients of signaling molecules and/or transcription factors and highlight the importance of studying this interplay in vivo (Miles, 2015).
To determine the molecular link between LSD1 and the Pumilio complex, tests were performed to see whether LSD1 can modulate the Pumilio complex expression. By conducting LSD1 ChIP from Drosophila and multiple mammalian cell lines, it was found that LSD1 is bound to the promoters of all of the components of the PUM complex. Although the binding of LSD1 to these promoters is conserved throughout evolution, LSD1's function in regulating PUM complex expression appears different in Drosophila and humans. Specifically, it was found that LSD1 acts as a transcriptional repressor of the NANOS genes in the human cells tested. In contrast, the results in Drosophila suggest that dLsd1 functions to promote rather than repress Nanos and Brat expression. It cannot be excluded that in complex tissues in Drosophila, dLsd1 depletion might indirectly cause Nanos and Brat down-regulation by altering tissue differentiation or by changing the expression of Pumilio complex transcriptional regulators. However, a dual role for LSD1 in controlling gene expression is consistent with previous studies showing that LSD1 can associate with both repressive (e.g., coREST), and activating co-factors, (e.g., Androgen receptor), to modulate gene expression. Intriguingly, some of the genes of the Pumilio complex, which are bound by LSD1, show only minor expression changes upon LSD1/2 depletion or inhibition (PUM1, PUM2). Double depletion of LSD1 and LSD2 seems to exclude that the lack of effect might be due to the possibility that LSD1 532 and LSD2 functions are redundant. Another possibility would be that LSD1 catalytic activity at these genes is blocked by specific co-factors or by the presence of acetylated histones, as previously observed in embryonic stem cells. Therefore, LSD1 may be bound to the actively transcribed Pumilio genes and be able to prime them for repression rather than directly contributing to their transcriptional potential. The current results highlight the importance of LSD1 in modulating the expression of the PUM complex but also suggest that this regulation is very context dependent (Miles, 2015).
In addition, this study also identified a conserved feedback mechanism between the PUM complex and LSD1 family members. The results demonstrate that the PUM complex directly targets the dLsd1/LSD2 transcripts and prevents their translation. It does this by binding to a NANOS regulatory element (NRE) within each of the 3'UTRs. Using luciferase reporter constructs, this study characterized a functional NRE motif within dLsd1 and LSD2. Consistently, dLsd1 and LSD2 protein levels were found to be sensitive to Pumilio manipulation. The result show that PUM does not affect LSD2 mRNA levels suggesting that PUM acts to directly inhibit the translation of LSD2 rather than promoting the degradation of the LSD2 transcript. It is proposed that the regulation of LSD2 and dLsd1 by the Pumilio complex represents a novel post-transcriptional regulatory mechanism to control the expression of these genes in specific cell types. The lack of PUM-binding sites in the shortened 3'UTR of LSD1 could allow for other ways to regulate LSD1, thus differentiating LSD1 and LSD2. For example, previous studies have identified additional post-transcriptional mechanisms, such as miR137, in constraining LSD1 levels. These results suggest that in human cells, the translation of both KDM1 family members are tightly regulated by different components of the post-transcriptional network (Miles, 2015).
Taken together the results suggest that dLsd1 and the Pumilio complex function with a built-in feedback loop which is important for tissue homeostasis during Drosophila development. Coupling Pum and dLsd1 expression may be a safeguard mechanism to control cell fates in a number of developmental contexts. Consistently, concomitant depletion of dLsd1 and the Pumilio complex were shown toresults in defects in wing vein determination. This interplay may also be important for oogenesis. In the Drosophila ovary, two independent Pumilio complexes function to regulate the balance between germ-line stem cell (GSC) self-renewal and differentiation into cystoblasts during oogenesis. The Nanos-Pumilio translational repressor complex is expressed in GSCs and promotes GSC self-renewal. In cystoblasts, a Brat-Pumilio complex functions to support differentiation. Importantly, dLsd1 mutant ovaries have an increased number of GSC-like cells and display a stem cell tumor phenotype. It is proposed that mis-regulation of Nos, Brat and Bam in dLsd1 mutants ovaries may contribute to the failure of stem cells to correctly differentiate into cystoblasts. These partially differentiated precursor cells accumulate in the ovary and generate stem cell tumors associated with dLsd1 loss. In mammalian systems, the family of LSD1 demethylases and the PUM complex act in an antagonistic manner. These findings have important ramifications for tumors, as LSD1 amplifications have been identified in a number of different tumor types. In tumors that over-express LSD1, such as bladder carcinoma, one might predict that LSD1 represses the expression of the PUM complex. Consistently, in the analysis of bladder carcinoma tumors, this study found significantly lower levels of NANOS1 and reduced levels of both PUM homologs (PUM1 and PUM2). Diminishing the levels of the PUM complex is likely to promote the translation of NRE-containing transcripts, including LSD2 and may contribute to the cellular changes associated with LSD1 amplifications. In support of this hypothesis, the over-expression of miRNAs targeting the PUM complex have been linked to aberrant expression of PUM substrates and to the progression of non-small cell lung tumors (Miles, 2015).
Based on these results, it is proposed that the LSD1 family of histone demethylases and the PUM post-transcriptional repressor complex are functionally linked in multiple organisms. These proteins are involved in intricate feedback loops during specific developmental contexts, which are likely to have important implications for stem cell biology and human cancers. These findings provide unexpected insights into the physiological consequences of altering epigenetic and post-transcriptional regulatory pathways and open the road to a detailed study of their impact on the balance between stem-cell renewal and differentiation (Miles, 2015).
Search PubMed for articles about Suppressor of variegation 3-3
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Chen, Y., et al. (2006). Crystal structure of human histone lysine-specific demethylase 1 (LSD1), Proc. Natl. Acad. Sci. 103: 13956-13961. PubMed ID: 16956976
Curtis, B. J., Zraly, C. B., Marenda, D. R. and Dingwall, A. K. (2011). Histone lysine demethylases function as co-repressors of SWI/SNF remodeling activities during Drosophila wing development. Dev Biol 350: 534-547. PubMed ID:21146519
Curtis, B. J., Zraly, C. B. and Dingwall, A. K. (2013). Drosophila LSD1-CoREST demethylase complex regulates DPP/TGFbeta signaling during wing development. Genesis 51: 16-31. PubMed ID:22965777
Dallman, J. E., Allopenna, J., Bassett, A., Travers, A. and Mandel, G. (2004). A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation. J Neurosci 24: 7186-7193. PubMed ID:15306652
Dietz, K. N., Di Stefano, L., Maher, R. C., Zhu, H., MacDonald, M. E., Gusella, J. F. and Walker, J. A. (2015). The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25168387
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. PubMed ID: 17462898
Di Stefano, L., et al. (2011). Functional antagonism between histone H3K4 demethylases in vivo. Genes Dev. 25(1): 17-28. PubMed ID: 21205864
Fuentes, P., Canovas, J., Berndt, F. A., Noctor, S. C. and Kukuljan, M. (2012). CoREST/LSD1 control the development of pyramidal cortical neurons. Cereb Cortex 22: 1431-1441. PubMed ID:21878487
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. PubMed ID: 12032298
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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. PubMed ID: 16079794
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Metzger, E., et al. (2005). LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437: 436-439. PubMed ID: 16079795
Miles, W.O., Lepesant, J.M., Bourdeaux, J., Texier, M., Kerenyi, M.A., Nakakido, M., Hamamoto, R., Orkin, S.H., Dyson, N.J. and Di Stefano, L. (2015). The LSD1 family of histone demethylases and the PUMILIO post-transcriptional repressor function in a complex regulatory feedback loop. Mol Cell Biol [Epub ahead of print]. PubMed ID: 26438601
Mulligan, P., Yang, F., Di Stefano, L., Ji, J. Y., Ouyang, J., Nishikawa, J. L., Toiber, D., Kulkarni, M., Wang, Q., Najafi-Shoushtari, S. H., Mostoslavsky, R., Gygi, S. P., Gill, G., Dyson, N. J. and Naar, A. M. (2011). A SIRT1-LSD1 corepressor complex regulates Notch target gene expression and development. Mol Cell 42: 689-699. PubMed ID:21596603
Ooi, L., et al, (2006). BRG1 chromatin remodeling activity is required for efficient chromatin binding by repressor element 1-silencing transcription factor (REST) and facilitates REST-mediated repression. J Biol Chem 281: 38974-38980. PubMed ID: 17023429
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. PubMed ID: 6819429
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date revised: 10 November 2013
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