InteractiveFly: GeneBrief

eggless: Biological Overview | References

BIOLOGICAL OVERVIEW

SET domain proteins are histone lysine methyltransferases (HMTs) that play essential roles in development. Histone methylation occurs in both the germ cells and somatic cells of the Drosophila ovary. The product of the eggless (egg) gene, an HMT, is required for oogenesis. Egg is a SET domain protein that is similar to the human protein SETDB1 and its mouse ortholog ESET. These proteins are members of a small family of HMTs that contain bifurcated SET domains. Because depletion of SETDB1 in tissue culture cells is cell-lethal, and an ESET mutation causes very early periimplantation embryonic arrest, the role of SETDB1/ESET in development has proven difficult to address. This study shows that egg is required in the Drosophila ovary for trimethylation of histone H3 at its K9 residue. In females bearing an egg allele that deletes the SET domain, oogenesis arrests at early stages. This arrest is accompanied by reduced proliferation of somatic cells required for egg chamber formation, and by apoptosis in both germ and somatic cell populations. It is proposed that other closely related proteins may function similarly in gametogenesis in other species (Clough, 2007).

Histones are subject to extensive post-translational modifications. The best studied of these histone modifications include the methylation of arginine (R) and lysine (K) residues, acetylation of K residues and phosphorylation of Serine (S) and Threonine (T) residues. The nucleosomal diversity created by these modifications forms the basis of the 'histone code' hypothesis, which proposes that chromatin domains, defined by local combinatorial signatures of histone modifications, have distinct outcomes for chromatin structure and gene expression (Clough, 2007).

Of the various modifications made to histones, methylation is one of the most complex, both in terms of the nature of the signal and its biological consequences. Both the site of methylation and the degree of methylation affect the biological outcome of methylation. For instance, histone H3 methylation at H3K4 (lysine at residue 4 in histone H3), H3K36 and H3K79 is usually associated with gene activation, whereas methylation at H3K9 and H3K27 leads to gene repression, although exceptions to these rules exist. Furthermore, K residues can be subject to different degrees of methylation (mono-, di- or trimethylation), with different consequences for the cell. These methyl marks serve as binding sites for proteins that assemble complexes that in turn regulate chromatin structure and gene transcription (Clough, 2007).

In 2000, the first histone lysine methyltransferase (HMT) was identified, Suv39h1, and its enzymatic activity was mapped to its SET domain. The SET domain takes its name from the three Drosophila genes in which it was first recognized: Su(Var)3-9, Enhancer of zeste (E(z)), and trithorax (trx). SET domain proteins have been identified and studied in yeast, plants, worms, flies and mammals. These proteins play important roles in development, and misexpression of HMTs occurs in some human cancers (Clough, 2007).

Gene silencing in germ cells is a widespread phenomenon, and recent studies have begun to examine the contributions of histone modifications to germ cell gene regulation. In Caenorhabditis elegans and Drosophila, formation of the embryonic primordial germ cells is accompanied by changes in histone acetylation and methylation, and two maternally expressed SET domain proteins, MES-2 and MES-4, are required in C. elegans for the viability and proliferation of the germline. Less is known about histone modifications in the adult gonad. This study shows that a SET domain protein, the product of a Drosophila gene that has been named eggless (egg), is required for oogenesis at early stages of egg chamber formation (Clough, 2007).

Egg is very similar to the human protein SETDB1 and its mouse ortholog ESET (Schultz, 2002; Yang, 2002). These proteins belong to a small subfamily of HMTs that contain bifurcated SET domains -- that is, SET domains interrupted by insertions of novel stretches of amino acids. The egg mutations that have been isolated have provided the opportunity to study the role of this subfamily of HMTs in a developmental context. This study shows in vivo that Egg catalyzes trimethylation of histone H3 at its K9 residue (H3K9), and that this modification is present in both germ and somatic cells during oogenesis. In the absence of Egg-catalyzed histone methylation, oogenesis arrests at early stages. Egg chamber formation is defective, and egg chambers never fully bud off from the germarium. This study shows that apoptotic cell death and reduced somatic cell proliferation are likely underlying causes of this early arrest (Clough, 2007).

The Drosophila gene eggless (egg) was identified in a genetic screen for EMS-induced lethal and female-sterile mutations uncovered by Df(2R)Dll-Mp. All 13 egg mutations isolated in this screen are female-sterile. In females bearing strong alleles (8), oogenesis is arrested at early stages, while weak alleles (5) are associated with mid-late stage oogenesis arrest. Some alleles also reduce viability, indicating that egg has other developmental functions in addition to its role in oogenesis. Rescue experiments, using DNA fragments from a region of DNA contained within the Df(2R)Dll-Mp deficiency, identified a 9 kb DNA fragment that contains the egg gene, and the position of egg within this fragment was mapped more precisely using a set of truncated and internally deleted fragments. Probes made from genomic DNA from this region hybridized to a single 3.9 kb band on Northern blots of poly(A+) RNA from adult ovaries. Using these probes, three overlapping cDNAs were isolated from a Drosophila ovary cDNA library. Sequence analysis of these cDNAs, as well as two embryo cDNAs obtained from the DGRC, yielded the proposed egg transcript structure. The Berkeley Drosophila Genome Project (BDGP) annotation of this region of the genome (version 4.3) predicted two genes, CG30422 and CG30426, but analysis of the above cDNAs, including one that is full length, shows the correct gene structure (Clough, 2007).

Egg contains a bifurcated SET domain, a methyl DNA-binding domain (MBD), and two Tudor-like domains. The SET domain is the catalytic domain of a family of HMTs that methylate histones on lysine residues. Egg also contains characteristic cysteine-rich Pre- and Post-SET domain regions that are necessary for enzymatic activity. Egg is the sole Drosophila protein to contain a bifurcated SET domain, an unusual disruption of the SET domain that characterizes its closest relative, human/murine SETDB1/ESET (Schultz, 2002; Yang, 2002). C. elegans also has a bifurcated SET domain protein, MET-2, of unknown function. Based on sequence comparisons, the SET domain of Egg falls into the Su(var)3-9 family of SET domains, which have specificity for methylating the K9 residue of histone H3 (Clough, 2007).

The predicted length of Egg (1262 amino acids) is close in size to SETDB1 (1291 amino acids) and ESET (1307 amino acids). A comparison of Egg and SETDB1 revealed that these proteins are 44% identical in the pre-SET plus first half of the SET domain, and 76% identical in the second half of the SET domain plus the post-SET domain. The MBD domains are less conserved (29% identical). While the putative Tudor domains in Egg were predicted with subthreshold E-values by the SMART protein domain identification program, the sequence similarity with SETDB1 in this region is high (44% identical). The amino acid segment that divides the SET domain of Egg is much shorter than the segment that interrupts the SET domain of SETDB1 or ESET (91 amino acids in Egg compared to 338 in SETDB1 and 337 in ESET) but is close in size to the corresponding segment of MET-2 (103 amino acids) (Clough, 2007).

A polyclonal antibody labels two protein bands on ovary western blots, ~170 kDa and ~140 kDa. Both proteins were absent or strongly reduced in egg²³⁵ ovaries, replaced by a smaller truncated protein, indicating that both are egg products. This antibody was used to label ovaries to determine the expression pattern and subcellular distribution of Egg during oogenesis (Clough, 2007).

Drosophila ovaries consist of 15-20 ovarioles that hold egg chambers in progressive stages of development. These egg chambers contain germ cells derived from germ stem cells located in the germarium, a structure at the tip of each ovariole. At their anterior ends (region 1), each germarium houses two to three germ stem cells (GSCs) that divide asymmetrically to produce another GSC, which replenishes the GSC population, and a cystoblast (CB). The CB undergoes four incomplete mitotic divisions (the dividing cells are called cystocytes) to yield a 16-cell germline cyst; one of these 16 germ cells becomes the oocyte, and the other 15 germ cells form the nurse cells. After germline cysts are formed, they become encapsulated (in region 2 of the germarium) by somatic prefollicular cells to form an egg chamber. At the posterior end of the germarium (region 3) lies a single stage 1 egg chamber, ready to bud off from the germarium and proceed through the rest of oogenesis (Clough, 2007).

Egg is expressed most strongly at early stages of oogenesis, in germ cells in the germarium. In germ stem cells and dividing germline cyst cells, the protein is present at roughly equal levels in the cytoplasm and the nucleus, but after 16 cell germline cysts have formed Egg accumulates preferentially in the nucleus. Egg is not detected in somatic cells at the tip of the germarium, including the terminal filament, inner sheath cells, or cap cells, but the protein is present at low levels in somatic cells in regions 2 and 3 of the germarium, including the prefollicular cells and the follicle cells of stage 1 egg chambers. Soon after egg chambers bud off the germarium, Egg levels increases in the follicle cells. By mid-oogenesis Egg decreases in the nurse cells, while protein levels continue to increase in the follicle cells. In the oocyte nucleus, Egg is present throughout the nucleoplasm and also localized to one to three distinct subnuclear sites associated with the chromosomes (Clough, 2007).

Experiments with tissue culture cells bearing reporter genes have shown that SETDB1 and ESET negatively regulate gene expression, and that their targets are likely to be euchromatic genes (Schultz, 2002; Yang, 2003). Despite these important studies, the biological roles of SETDB1 and ESET in development are poorly understood. RNAi knockdown of SETDB1 in tissue culture cells is cell lethal (Wang, 2003; Sarraf, 2004), and the very early embryonic lethality of a mouse ESET insertion allele, along with possible maternal contributions of its gene product, has complicated the analysis of its in vivo functions (Dodge, 2004). The genetic analysis of egg reported here has allowed examination of the in vivo contributions of this HMT in a developmental context, and to show that Egg plays an essential role in oogenesis. The strong expression of ESET in testes suggests that the mammalian proteins may also play similar roles in gametogenesis (Yang, 2002) (Clough, 2007).

Biochemical studies demonstrated that ESET and SETDB1 methylate histone H3 at its K9 residue (Schultz, 2002; Yang, 2002; Wang, 2003; Yang, 2003), and this study shows that in vivo Egg also has H3K9 HMT activity. Specifically, it was found that trimethylation of histone H3K9 occurs during oogenesis, in both the germ cells and somatic cells, in an egg-dependent manner. Egg does not appear to be required for dimethylation of H3K9, since the H3K9me2 signal remained strong in egg¹⁴⁷³ ovaries. These observations suggest a pathway for H3K9 methylation, with Egg catalyzing the addition of a terminal methyl group to H3K9me2, previously established by a separate HMT (Clough, 2007).

Egg is present in germ cells at the earliest stages of oogenesis, including germ stem cells, cystoblasts, dividing cystocytes and newly formed germline cysts. While Egg was not detected in anterior somatic cells, including terminal filament, cap and interstitial cells, low levels of Egg were present in more posterior somatic cells, including prefollicular cells and follicle cells of stage 1 egg chambers. However, Egg was also expressed in postgermarial egg chambers, and is therefore likely to have functions at later stages of oogenesis as well. Of particular interest is the strong accumulation of Egg in the oocyte nucleus, in distinct subnuclear foci. The oocyte nucleus is arrested at prophase of meiosis I, and is generally transcriptionally quiescent, raising the possibility that Egg could contribute to transcriptional repression in the oocyte and/or meiotic cell cycle control (Clough, 2007).

Strong egg alleles, including egg¹⁴⁷³, which deletes the entire SET-domain-coding region, causes very early arrest of oogenesis. Mutant ovaries consist of germaria in which the early stages of egg chamber formation are not clearly demarcated. While proliferating germ cells are present, the existing germline cysts were not fully encapsulated by somatic follicle cells, and did not bud off normally from the germarium (Clough, 2007).

egg is required for the proliferation and viability of somatic cells in the germarium, and a reduction in somatic cell populations is likely to be the cause of the encapsulation and budding defects observed in mutant germaria. In wild-type germaria, mitosis was observed in prefollicular and follicle cell populations, as well as in single cells located near the 2a/b border, where somatic stem cells reside. Since the only examples of somatic cells undergoing mitosis in egg¹⁴⁷³ ovaries are cells positioned at the posterior end of germaria, it is likely that egg affects proliferation of at least three populations of somatic cells: the somatic stem cells, the prefollicular cells and the follicle cells that surround newly formed egg chambers. egg is also required for the viability of both the germ and somatic cells, since apoptotic cell death occurs in both cell types in egg¹⁴⁷³ ovaries (Clough, 2007).

Several HMTs play roles in cell proliferation, and aberrant HMT expression is in some cases oncogenic. Histone methylation can impinge on cell proliferation by either of two routes: by regulating the expression of genes that in turn regulate the cell cycle, or by promoting structural changes in chromosomes necessary for mitosis. SETDB1 has recently been shown (Li, 2006) to function at promoters that are silenced in human cancers, suggesting that it too may normally play a role in regulating cell proliferation (Clough, 2007).

There are important questions that remain to be answered. Egg mediates H3K9 methylation in early oogenesis, and that loss of H3K9me3 has striking biological consequences for oogenesis, but the exact genomic effects of this methylation program are not yet known. Methylation of H3K9 plays an important role in the formation of heterochromatin domains, and also regulates the expression of individual euchromatic genes. Analysis of Egg localization in whole-mount ovaries indicates that it is associated with distinct foci within germ and somatic cell nuclei, but the small size of these chromosomes has not allowed these sites to be precisely mapped. A goal of future work will be to identify the genomic targets of Egg, a necessary and important first step in determining whether Egg regulates euchromatic gene expression or plays a role in establishing heterochromatic domains (Clough, 2007).

Another important goal of future work is to determine which cell types require egg activity during early oogenesis. While it has been shown that Egg is expressed in both germ cell and somatic cell populations in the ovary, and mediates H3K9 methylation in both cell types, the fact that numerous germ cell-somatic cell interactions contribute to early oogenesis implies that the functional consequences of perturbations in histone methylation patterns in one cell type could impact on the development of other cells. Thus somatic cell defects could arise from loss of H3K9me3 in germ cells, and vice versa. Future experiments, using clonal analysis and the expression of egg transgenes in specific cell types, should allow determination unequivocally of which cells require the HMT activity of egg (Clough, 2007).

Drosophila SETDB1 is required for chromosome 4 silencing

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3–9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. This study shows that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing (Seum, 2007).

Whereas Su(var)3–9 and dG9a are not essential, DmSetdb1 is the first gene described encoding a H3K9 methyltransferase that is required for fly viability. DmSetdb1 transcript can be detected at every stage of development. Analysis by Northern blot confirms that the only transcript is 3.9 kb long, encompassing both CG30422 and CG30426. Early embryos show relative high mRNA levels, suggesting deposition of the transcript in the embryo. Others have reported that DmSetdb1 transcript is not present in 0–3-h embryos when tested by RT-PCR, a result that is not easily reconciled with the observations. DmSetdb1^10.1a homozygotes are rescued with the UAS- DmSetdb1^421–1,261 daGal4 transgene; the males are fertile, while the females are sterile. Thus, the rescue is not complete in females, because of either nonappropriate expression of the transgene or because DmSETDB1^421–1,261 is not full-length. This observation is consistent with the fact that DmSetdb1 (eggless) has been shown to be required for oogenesis. Preliminary data suggest that sterility in rescued females and in eggless mutant alleles is due, at least in part, to defects in germline development. Indeed, using the FLP-ovo^D1 system, no DmSetdb1^10.1a homozygous mutant germline clones could be generated. This suggests that germline-specific expression of DmSetdb1 is required before stage 5 of oogenesis. This does not exclude, however, that a maternal contribution is required for proper oogenesis (Seum, 2007).

The polyclonal antibody directed against a DmSETDB1 peptide that was generated does not recognize DmSETDB1 on polytene chromosomes. Therefore, the DmSetdb1^3HA allele was generated that results in the expression of the endogenous DmSETDB1 protein tagged with 3HA. Such an approach has the advantage that the endogenously expressed protein can be detected with highly specific monocolonal antibodies. This showed that DmSETDB1 localizes at a high level on Chromosome 4 and over the chromosome arms. DmSETDB1 is also present at the chromocenter. It is not known if this feature has any biological significance as DmSETDB1 does not methylate the chromocenter. The association of DmSETDB1 with chromatin is not dependent on its own catalytic activity, since the DmSETDB1^421–1,261(H1195K) mutant protein localizes similarly to DmSETDB1^421–1,261. The mode of DmSETDB1 recruitment thus differs from that of SU(VAR)3–9, since the latter appears to require its HMTase activity for binding to heterochromatin. It is currently not known how DmSETDB1 is recruited to chromatin. Mammalian SETDB1 is recruited to DNA together with HP1, either via the KRAB-zinc-finger protein KAP1 corepressor, or as a component of the MBD1-mAM/MCAF1-SETDB1 complex. It is tempting to speculate that in Drosophila transcriptional repressors also recruit DmSETDB1 onto euchromatin or at Chromosome 4 (Seum, 2007).

Comparative analysis of H3K9 methylation and HP1 profile on polytene chromosomes of wild-type and DmSetdb1^10.1a homozygous mutant larvae shows that DmSETDB1 is involved in some of the H3K9 mono- and dimethyl marks in euchromatin and in dimethyl marks on Chromosome 4. Loss of methylation at Chromosome 4 and euchromatin is coherent with the localization profile of the DmSETDB1 protein itself. Western blot analysis of the H3K9 methylation level in mixed salivary glands, brain, and imaginal discs tissue in DmSetdb1 mutant background shows a decrease in all three H3K9 methyl marks. No change of trimethylation in polytene chromosomes of DmSetdb1^10.1a mutant larvae was evident. This suggests a distinct H3K9 trimethylation profile in the tissues analyzed by Western blot and in polytene chromosomes. This hypothesis is corroborated by the recent finding that DmSETDB1 trimethylates H3K9 in germ and somatic cells of the germarium (Seum, 2007).

The overexpression data provide a mirror image, in that they show the ability of DmSETDB1 to mono-, di-, and trimethylate H3K9. Thus, Drosophila DmSETDB1 and mammalian SETDB1 are conserved with respect to their HMTase activity, as both Drosophila DmSETDB1 and mammalian SETDB1 are H3K9 mono-, di-, and tri-HMTases. Although such a mechanism has not yet been described, it cannot be excluded that DmSETDB1 is exclusively a H3K9 monomethyltransferase providing monomethyl substrates for other enzymes; but in that case, the partner enzyme would not be SU(VAR)3–9, since its absence does not impair Chromosome 4 or euchromatic dimethylation. In mammals, conversion of the H3K9 dimethyl- to the trimethyl-state by SETDB1 is strongly facilitated by the mAM cofactor. Such a mechanism can also be envisaged for DmSETDB1, and CG12340 is a candidate Drosophila homologue of mAM (Seum, 2007).

No HMTase activity of DmSETDB1 could be detected in cell-free conditions. Immunopurified DmSETDB1, regardless of whether expressed in mammalian or in Drosophila S2 embryo cell lines, did not show any activity when tested on GST-H3, GST-H4, core histones, or oligonucleosomes, while mammalian SETDB1 produced under identical conditions showed robust H3 specific activity. It is hypothesized that another protein or a post-translational modification is necessary for HMTase function of DmSETDB1. This activity would not be present in S2 cell line; this is consistent with the fact that overexpression of DmSETDB1 in S2 cells does not induce any increase in H3K9 mono-, di-, or trimethylation (unpublished data) (Seum, 2007).

DmSETDB1 functions in association with HP1; HP1 is recruited when DmSETDB1^421–1,261 is overexpressed and lost from some euchromatic bands and Chromosome 4 in the DmSetdb1^10.1a mutant. In addition, HP1 is required for DmSETDB1-dependent repression of Chromosome 4 variegating transgenes. It is speculated that HP1 is recruited to chromatin by both the DmSETDB1 protein and the H3K9 methyl mark. Indeed, the DmSETDB1 protein is not able to recruit HP1, because the DmSETDB1^421–1,261(H1195K) mutant protein does not influence HP1 localization. On the other hand, the H3K9 methyl mark alone is not sufficient to recruit HP1. Therefore, it is hypothesized that HP1 recognizes the H3K9 methyl mark in association with DmSETDB1, or with another factor. The situation is similar for Suv39H1, where the protein itself does not recruit HP1, despite a direct interaction that is necessary for HP1 binding in collaboration with the H3K9 methyl mark. It is not known if a direct DmSETDB1-HP1 interaction occurs, but two arguments in mammals argue in favor of this. First, KAP1 directly binds HP1 and SETDB1, and in such a complex, contacts between HP1 and SETDB1 are probable. Second, heterochromatin targeted HP1 recruits SETDB1, although an intermediate factor cannot be excluded (Seum, 2007).

Although both DmSETDB1 and SU(VAR)3–9 methylate H3K9, one cannot substitute for the other. Indeed, in a mutant background for one enzyme, the other will not compensate for its absence. In addition, both enzymes function independently; SU(VAR)3–9-mediated H3K9 di- and trimethylation and HP1 deposition at the chromocenter are not affected in the DmSetdb1^10.1a mutant context, and conversely, H3K9 mono- and dimethyl marks at euchromatic arms, dimethyl marks on Chromosome 4, and the associated HP1, are not affected in a Su(var)3–9 mutant background. Surprisingly, SU(VAR)3–9 is present on Chromosome 4; it is most probably recruited by HP1, but it does not induce any H3K9 methylation. Thus, DmSETDB1 and SU(VAR)3–9 exert nonoverlapping and independent functions, suggesting that they accomplish distinct biological roles. It is anticipated that at least one additional HMTase is involved in H3K9 methylation in Drosophila. H3K9 monomethylation at the chromocenter, H3K9 dimethylation at the telomeres, and some of the H3K9 mono- and dimethylation marks at euchromatic bands are not deposited by SU(VAR)3–9 nor DmSETDB1. One candidate, dG9a, was recently shown to methylate H3K9 and to localize to euchromatin (Seum, 2007).

The repressive function of DmSETDB1 demonstrated for Chromosome 4 is consistent with the fact that H3K9 methylation is generally found in association with transcriptional silencing. Indeed, the mammalian SETDB1 homologue fulfills such a function. DmSETDB1 could also be implicated positively in gene expression, since H3K9 di- and trimethylation, as well as HP1γ were recently found in the coding region of active genes. One task will be to identify endogenous genes that are regulated by DmSETDB1 in euchromatin and at Chromosome 4. Genes located in the region 31 are potential candidates, given that the HP1 signal is lost in the DmSetdb1^10.1a mutant. The second set of candidate genes are those physically associated with HP1 but not with SU(VAR)3–9. Large-scale mapping of HP1 and SU(VAR)3–9 targeted loci in embryonic Kc cells has shown that whereas HP1 and SU(VAR)3–9 bind together to transposable elements and pericentric genes, HP1 binds to many genes on Chromosome 4, mostly independently of SU(VAR)3–9. The latter, together with a class of euchromatic genes showing the same protein-factor occupation profile, possibly depend on DmSETDB1 for H3K9 methylation and regulation (Seum, 2007).

DmSETDB1 is the H3K9 HMTase responsible for heterochromatin silencing on Chromosome 4, because variegating transgenes are derepressed in a DmSetdb1^10.1a mutant background. As both alleles have to be mutated in order to obtain an effect, the DmSetdb1 gene is a recessive suppressor of variegation on Chromosome 4. Conversely, loss of a single dose of HP1 or SU(VAR)3–7 results in loss of silencing. This difference could be explained by the fact that DmSETDB1 is an enzyme, whereas HP1 and SU(VAR)3–7 are dosage-sensitive structural components. Alternatively, DmSETDB1 might be present in excess. Heterochromatic variegating reporters are responding to an additional or missing dose of SU(VAR)3–9 when inserted on Chromosomes 2, 3, or X, but not on Chromosome 4. This observation is henceforth explained by the fact that DmSETDB1 mediates H3K9 dimethylation on Chromosome 4. Conversely, and as expected, variegating expression responding to the SU(VAR)3–9 dosage is not under the control of DmSETDB1. This corroborates once again that SU(VAR)3–9 and DmSETDB1 function independently. Mammalian SETDB1 is involved in epigenetic maintenance, since silencing is stably maintained for more than 40 population doublings, once it is established on an integrated reporter by a short transient pulse of the corepressor KAP1 that subsequently recruits SETDB1 and HP1. DmSETDB1 could also be involved in epigenetic maintenance; in that case, transient expression would suffice for long-term repression of Chromosome 4 variegating transgenes (Seum, 2007).

The arm of Chromosome 4 is composed of a minimum of three euchromatic domains interspersed with heterochromatic domains. The variegating P elements that were tested were inserted within the banded region, in or at the edge of heterochromatic domains. Chromosome 4 heterochromatic bands are qualitatively different from centromeric heterochromatin, as they are H3K9 dimethylated and regulated by DmSETDB1, not by SU(VAR)3–9. Two possibilities can be envisaged for the Chromosome 4 domains that are methylated by DmSETDB1. First, they could be representative of equivalent bands at euchromatic arms, which would be smaller and/or more dispersed, and therefore would not yet have been identified functionally. Alternatively, D. melanogaster Chromosome 4 could make use of specific machinery dedicated to gene regulation and/or epigenetic maintenance. The other well-known example of chromosome-specific regulation is the dosage compensation of sex chromosomes. In that case, DmSETDB1 function would depend on partners or DNA sequences specific for Chromosome 4, such as for instance the Chromosome 4-specific factor POF or the Hoppel element, also known as 1360, which is over-represented on the D. melanogaster Chromosome 4, and which could be an initiation site for heterochromatin formation (Seum, 2007).

In conclusion, this study has characterized DmSETDB1 as a major nonheterochromatic H3K9 methyltransferase in Drosophila. It was also demonstrated that DmSetdb1 is an essential gene and that its loss has functional consequences on gene expression on Chromosome 4. This work represents an important step toward the understanding of the differential specificity and mode of action of distinct H3K9 HMTases and underlines a specific mode of regulation of Chromosome 4 in Drosophila (Seum, 2007).

dSETDB1 and SU(VAR)3–9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true 'immortal stem cells'. In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. This study shows that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3–9. Loss-of-function mutations in dsetdb1 (eggless) or Su(var)3–9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3–9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype (Yoon, 2008).

This study shows that dSETDB1 is the only HKMTase responsible for the synthesis of H3K9me3 signals in the inner germarium where GSCs and their early descendants are found. When these vasa-positive cells move to region-3 germarium, the H3-K9 trimethylating task is transferred to a combination of dSETDB1 and SU(VAR)3–9, as both enzymes act cooperatively in all other somatic-type cells of the germarium. After the egg chamber buds off from the germarium, the trimethylation activity is now entirely the province of SU(VAR)3–9. The results disclose that the developmental program uses dSETDB1 first and then SU(VAR)3–9 during GSC differentiation, indicating that the two HKMTases perform distinct functions in these germ cells. The role of dSETDB1 in early GSC differentiation is presumably to 'pre-mark' certain regions of chromatin, including the pericentric heterochromatin, with H3K9me3. The biochemical features of these pre-marked regions might be different from those of regions that are substrates of SU(VAR)3–9, and the pre-marked regions may be the platform on which incoming SU(VAR)3–9 further modulates the pre-methylated chromatin regions in later-developing VASA-positive cells. The functional significance of trimethylating, or 'priming', GSC chromatins with dSETDB1 is highlighted by the catastrophic ovarian phenotypes observed in, and the sterility of, the dsetdb1 female homozygote. By contrast, although the egg chambers of the Su(var)3–9¹⁷ mutant completely lacked H3K9me3 signals, which might be expected to result in a phenotype more severe than that of the dsetdb1 mutant, the Su(var)3–9¹⁷ mutant is capable of oogenesis, is able to lay eggs, and is fertile (Yoon, 2008).

Meanwhile, the localization of both dSETDB1 and Su(var)3-9 at DAPI-dense heterochromatin does not necessarily mean that they target the same chromatin loci in early- and late-stage of oogenesis, respectively. The observation that dSETDB1, but not Su(VAR)3-9, is essential for Drosophila oogenesis provides a possibility that the two HKMTases may have different sets of target chromatin regions during oogenesis. It would be interesting to examine whether dsetdb1 phenotypes could be rescued or not if exogenous Su(var)3-9 were expressed at high level in GSCs and their close derivatives (Yoon, 2008).

In germ cells, dSETDB1 locates at DAPI-dense, pericentric heterochromatin. This was unexpected because the mammalian counterpart, SETDB1/Eset, is known to have euchromatin-associated function. Seum (2007) recently reported that, in Drosophila polytene chromosomes, dSETDB1 locates at the fourth chromosome. This fourth chromosome is known to be unusual as it has many characteristics of heterochromatic domains (such as a high-repeat density, no recombination and late replicating) and, at the same time, it shows features of euchromatin (such as being transcriptionally active and having a high gene density); in fact, many of the genes in the fourth chromosome are expressed during development. These characteristics indicate that the banded regions of the fourth chromosome are different from pericentric heterochromatin, which highlights the peculiarity of dSETDB1 localization to DAPI-dense heterochromatin in the germarium (Yoon, 2008).

The location of dSETDB1 at pericentric heterochromatin probably indicates that dSETDB1 participates, at a global level, in regulating chromosome organization and maintaining the chromosome integrity in the germ-lineages. This hypothesis is supported by the observation that the main H3K9me3 spots were displaced and went astray from DNA-dense heterochromatin regions in most region-3 cells of the dsetdb1^G19561 germarium. In addition, the egg chambers of the dsetdb1^G19561 mutant ovary that survived stage six were shown to have disorganized chromosomes in the nurse cell nuclei. The nurse cell chromosomes of the stage-7 egg chambers in both wild-type and Su(var)3–9¹⁷ ovaries were organized into bundles with well-developed, large nucleoli, whereas those in the dsetdb1^G19561 ovaries were simply scattered throughout the nucleoplasm without nucleolar regions; otherwise, all were stained positive in the TUNEL assay. HP1 was diffusely located in these nuclei of dsetdb1^G19561 mutant egg chambers but the HP1 mislocalization is unlikely to be the reason for the scattered chromosomes because the Su(var)3–9¹⁷ egg chambers totally lacked HP1 but had nucleolar regions between bundles of chromosomes. These results suggest that dSETDB1 has a role in coordinating the chromosomal integrity in the germ-cell lineages, and the loss of dSETDB1 function results in a dysregulation of chromosome organization (Yoon, 2008).

In the ovary, the main type of methylation catalyzed by dSETDB1 is H3K9me3. In the dsetdb1^G19561 germarium, the loss of H3K9me3 was limited to the germ cells in the inner germarium. By contrast, in the salivary glands, dSETDB1 primarily synthesizes H3K9me2 at the fourth chromosome at which dSETDB1 itself localizes. Alterations in the H3K9me3 pattern and intensity were not detected in the polytene chromosomes in these studies. This means that dsetdb1 synthesizes either H3K9me2 or H3K9me3, depending on the type of cells in which it functions. By analogy with mammalian SETDB1/Eset, dSETDB1 can produce in vitro all the methylation types such as H3K9me1, H3K9me2 and H3K9me3. Under in vivo conditions, the specificity of SETDB1 activity and the resulting state of methylation depend on regulatory protein(s) associated with SETDB1/Eset. This is shown by the observation that a murine ATFa-associated factor (mAM) tightly associates with SETDB1 and facilitates the SETDB1-dependent conversion of H3K9me2 to H3K9me3. Therefore, the proteins that regulate SETDB1 activity determine the H3-K9 methylation state in certain tissue cells and at particular developmental stages, and this might be true for dSETDB1 in Drosophila (Yoon, 2008).

Relating to likely dSETDB1-associated protein(s), a clue was provided by the observation that a dsetdb1 null mutant, DmSetdb1^10.1a, dies at the late pupal stage, but it can be rescued to progress to the adult stage by expression of a truncated DmSETDB1^421–1,261 transgene, which was constructed by deleting the N-terminal 420 amino acids of the full-length dSETDB1. Of particular interest is the finding that the rescued females are sterile whereas the males are fertile, which is the same phenotype as seen with the dsetdb1^G19561 mutant. This rescue experiment indicates that the truncated DmSETDB1^421–1,261 is enough for the null DmSetdb1^10.1a mutants to survive the pupal stage, but is still insufficient to overcome the female sterility. This provides important clues about the tissue and substrate specificity of dSETDB1. This indicates that the N-terminal region (spanning 1–420 amino acids) of dSETDB1 is instrumental in female fertility. This region likely forms a functional domain that provides a binding site(s) for regulatory protein(s) that positions dSETDB1 at pericentric heterochromatin in the PGCs and GSC-derived cells instead of the fourth chromosomes, and preferentially synthesizes H3K9me3 instead of H3K9me2. Mammalian SETDB1/ESET is known to associate with several transcriptional regulators such as the ERG protein, mAM, KRAB-zinc-finger protein KAP1, and MBD1/MCAF1. It would be interesting to investigate the factor(s) that restricts dSETDB1 to the germ-cell lineages and favors H3K9me3 over H3K9me2 in the ovary (Yoon, 2008).

At present, there is no information on SU(VAR)3-9 function during the Drosophila oogenesis. Because an antibody capable of immunocytochemically detecting SU(VAR)3-9 protein was unavailable, a transgenic line was used that expresses GFP-tagged SU(VAR)3-9 protein as an alternative. It is clear that the ectopic expression pattern shown by the GFP-tagged SU(VAR)3-9 does not always reflect the pattern of endogenous SU(VAR)3-9. Nevertheless, if the SU(VAR)3-9-eGFP were expressed in a cell with endogenous SU(VAR)3-9, the SU(VAR)3-9-eGFP signals would be localized to wherever endogenous SU(VAR)3-9 is located. The results of RISH and RT-PCR analyses showed that Su(var)3-9 is expressed in the ovarioles including the germarium and participates in oogenesis, and the egg chambers were shown to lack H3K9me3 in the Su(var)3–9¹⁷ mutant flies. Therefore, a GFP-tagged SU(VAR)3-9 transgenic fly was used to determine the location of endogenous SU(VAR)3-9 from the ectopically expressed SU(VAR)3-9-eGFP signals in the ovarian cells, and the results showed that the SU(VAR)3-9-eGFP signals were overlapped with H3K9me3/HP1 signals in the egg chambers, indicating that endogenous SU(VAR)3-9 is responsible for H3K9me3 signals in developing egg chambers. The function of SU(VAR)3-9 in the germarium could also be deduced from the localization of SU(VAR)3-9-eGFP signals. In the inner germarium SU(VAR)3-9-eGFP signals were less co-localized with H3K9me3 signals than in the outer germarium. Such a positioning of SU(VAR)3-9-eGFP in the germarium is in agreement with the prediction of endogenous SU(VAR)3-9 function in the outer germarium. Therefore, it is certain that SU(VAR)3-9 also has a role in the oogenesis. Despite its role as an influential epigenetic modifier, the SU(VAR)3-9 function during the oogenesis is likely to be dispensable because Su(var)3-9 null mutant flies are fertile (Yoon, 2008).

HP1 recognizes H3K9me2 and H3K9me3. In the polytene chromosomes of salivary glands, HP1 localizes at the chromocenter and chromosome 4, in agreement with the pattern of H3K9me2, rather than H3K9me3, which is present at the core of the chromocenter. Mutations in the dsetdb1 gene abolish both H3K9me2 and HP1 signals from the fourth chromosome in the salivary glands. By contrast, the HP1 in the nuclei of both the germarium and the developing egg chambers mainly associates with H3K9me3 instead of H3K9me2. The dsetdb1^G19561 germarium and the Su(var)3–9¹⁷ egg chambers have normal-looking H3K9me2 signals but lack H3K9me3, and their nuclei also lack HP1 signals. These observations indicate that in some cells and tissues, HP1 binds either H3K9me2 or H3K9me3, and the preferred substrate depends on the HKMTase(s) itself that recruits and tethers HP1 to their sites of action (Yoon, 2008).

In summary, this study has demonstrated that dsetdb1 is expressed, in a germ cell-specific manner, in the germarium; the germline stem cells and their early descendants reside in the anterior part of the germarium and both H3K9me3 and HP1 signals are abolished with mutations in the dsetdb1 gene. In the GSC-derived cells, dSETDB1 trimethylates H3-K9 residues at pericentric heterochromatin, but this function is performed by SU(VAR)3–9 as germline cysts differentiate into egg chambers. Loss-of-function mutation in Su(var)3–9 abolishes both H3K9me3 and HP1 signals in developing egg chambers. Both dSETDB1 and SU(VAR)3-9 collaborate in the region-3 germarium and a mutation in either of these genes causes localization of H3K9me3 away from DNA-dense regions in the region-3 cells. These findings, therefore, indicate that dsetdb1 and Su(var)3-9 act sequentially to regulate chromosome organization in accordance with the differentiation of the germline-stem cells in Drosophila (Yoon, 2008).

Windei, the Drosophila homolog of mAM/MCAF1, is an essential cofactor of the H3K9 methyl transferase dSETDB1/Eggless in germ line development

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. This study shows that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, the protein regions in Wde and Egg were identified that are necessary and sufficient for the interaction between the two proteins. A region of Egg was identified that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. These data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells (Koch, 2009).

This study analyzed the function of Wde, the Drosophila homolog of mAM/MCAF1, in development. Wde precisely colocalizes with Egg and the mutant phenotypes of wde and egg are indistinguishable, indicating that Wde is an indispensable binding partner of Egg required for trimethylation of H3K9 at euchromatic sites, in particular on the fourth chromosome. Functional data on mAM/MCAF1 have so far only been obtained by RNAi-mediated knock-down or by expression of mutated mAM/MCAF1 proteins in tissue culture cells. One study concluded that mAM/MCAF1 increases the enzymatic HMT activity of SETDB1, in particular with respect to the conversion of H3K9me2 to H3K9me3. A second study showed that expression of mAM/MCAF1 mutated in its binding site for MBD1 interferes with recruitment of SETDB1 to chromatin. The current study is the first using a null mutant of a mAM/MCAF1 homolog and the results clearly show the strict requirement for Wde for proper localization and in vivo function of Egg in germ line cells (Koch, 2009).

It has been shown that mAM/MCAF1 and SETDB1 associate in a protein complex and that a short region of mAM/MCAF1 including the coiled-coil domain is sufficient for binding to SETDB1. This study has confirmed and extended these observations by showing that a region including the coiled-coil domain of Wde is sufficient for binding to Egg and that a short region of Egg (aa 366–521) devoid of any known protein motif is sufficient for binding to Wde. For mAM/MCAF1 it was proposed that its binding to SETDB1 alters the catalytic activity and substrate specificity of the histone methyl transferase domain, thus allowing efficient trimethylation of H3K9. While the same may be true for the Wde/Egg interaction, the results show that in the absence of Wde, Egg is hardly detectable in germ line cells, most likely because Wde is required to protect Egg from proteolytic degradation. Moreover, when Egg is overexpressed in the absence of Wde, it fails to localize to the nucleus, revealing an additional function for Wde in nuclear import of Egg (Koch, 2009).

On polytene chromosomes Wde binds strongly to the fourth chromosome and to multiple euchromatic bands on all other chromosomes. Strong binding to the fourth chromosome has also been reported for Egg and is consistent with the hypothesis that Egg may be specifically required for euchromatic H3K9 trimethylation on the fourth chromosome, which is not affected in Su(var)3–9 and G9a mutants. Two recent studies showed indeed that Egg specifically affects the transcription of loci located on chromosome 4. However, the two studies come to apparently contradictory results. While the first study one reported derepression of transgenes inserted on chromosome 4 in egg mutants, the second study reported a general reduction of the transcription of genes on the fourth chromosome in egg mutants, measured in a microarray experiment. Nonetheless, the involvement of both Egg and Wde in the transcriptional regulation of genes on chromosome 4 appears very likely, since both Egg and Wde bind to POF. It could not be determine whether Wde and Egg bind to POF independently or sequentially, because it cannot be excluded that the expression of endogenous Egg in S2 cells contributes to the binding of transfected Wde and POF (Koch, 2009).

POF is a unique example of a protein that specifically associates with a single autosome, the fourth chromosome of Drosophila melanogaster. In pof mutants, the transcription level of genes on the fourth chromosome is reduced, indicating that POF promotes transcription of genes on chromosome 4. In contrast, the localization of POF to chromosome 4 is dependent on HP1 and vice versa, and there appears to be competition between these two proteins for binding to genes and their promoters on chromosome 4. These observations have led to the model that the activities of HP1 and POF have to be balanced in order to ensure transcription of genes on chromosome 4 at the right level. It is proposed that Egg and Wde are part of this balancing mechanism because both proteins bind to POF and recruit HP1 by generating H3K9me3 marks on chromosome 4 (Koch, 2009).

Two recent studies showed that Egg is required for the development of ovaries in Drosophila. Ovaries of homozygous egg mutant females are rudimentary and degenerate by apoptosis before egg chambers bud off the germarium. This result has been confirmed and this study has shown that homozygous wde mutant females show exactly the same phenotype. From these observations it was not clear whether the function of Egg and Wde is required in the germ line cells, the somatic follicle cells, or both. To address this question, the function of egg and wde in germ line cells was eliminated by FLP/FRT mediated mitotic recombination. Egg chambers with egg or wde germ line clones did develop up to stage 8 of oogenesis, but subsequently degenerated due to apoptosis. Because the ovary phenotype of homozygous mutant egg and wde females was more severe than the germ line clone phenotype of mutants in both genes, it is concluded that wde and egg may also be required for proper development of somatic follicle cells (Koch, 2009).

It has been speculated that Egg may be dispensable for trimethylation of H3K9 at later stages of oogenesis because this function could be taken over by Su(var)3–9 (Yoon, 2008). However, this hypothesis is not consistent with the different localization of the Wde/Egg complex and Su(var)3–9 on salivary gland polytene chromosomes and with the different consequences of the respective mutations on H3K9 methylation in pericentric heterochromatin and euchromatin, in particular on chromosome 4. Furthermore, mutations in wde and egg lead to apoptosis of germ line cells, which obviously cannot be rescued by the presence of Su(var)3–9 which is already expressed in the germ line at the time when apoptosis starts (Koch, 2009).

Modification by SUMOylation and binding to SUMO is a common hallmark of many chromatin regulators involved in transcriptional repression. Both mAM/MCAF1 and SETDB1 can bind SUMO and it has been suggested that this property is required for the recruitment of these proteins to promoters bound by transcriptional repressors such as KAP1, Sp3 and MBD1. The finding that Egg is itself modified by SUMOylation suggests that binding of additional chromatin modifiers to SUMOylated Egg may contribute to the efficient assembly of higher order chromatin repression complexes at specific euchromatic sites (Koch, 2009).

Histone H3K9 trimethylase Eggless controls germline stem cell maintenance and differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. This study shows that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms (Wang, 2011).

Although the mouse H3K9 trimethylase SETDB1 was recently shown to be important for maintaining ESC self-renewal by repressing the expression of developmentally regulated genes (Bilodeau, 2009), its role in regulation of adult stem cells has not yet been established. In this study, it was shown that the Drosophila SETDB1 homolog, Egg, is required intrinsically for controlling GSC self-renewal and extrinsically for controlling GSC differentiation in the Drosophila ovary. The egg mutant ovaries exhibit both GSC loss and germ cell differentiation defects. It was further demonstrated that Egg controls GSC self-renewal by repressing a Bam/Bgcn-independent pathway. In addition, escort cell-specific RNAi-mediated knockdown of egg function leads to gradual escort cells (EC) loss and germ cell differentiation defects, indicating that Egg is required for EC maintenance and germ cell differentiation. Recently, it has been proposed that ECs function as a niche for promoting germ cell differentiation (Kirilly, 2011). Furthermore, Egg functions in ECs to control germ cell differentiation at least in part by preventing BMP signaling from spreading to the differentiation niche and regulating EC survival. Therefore, it is proposed that Egg is a key H3K9 trimethylase in the Drosophila ovary that is required intrinsically for controlling GSC self-renewal via repressing a Bam/Bgcn-independent differentiation pathway and in ECs for controlling germ cell differentiation by preventing BMP signaling spreading to the differentiation niche. The findings from this study have further supported the idea that ECs function as a germ cell differentiation niche. It will be of great interest to test if SETDB1 is also important for controlling adult stem cell self-renewal and differentiation in mammalian systems (Wang, 2011).

A previous study has shown Egg to be a primary H3K9 trimethylase in follicle progenitor cells for maintaining H3K9me3 and regulating their proliferation and survival. Egg and its co-factor Wde were also shown to be required for maintaining H3K9me3 in early germ cells and regulating their survival. This study has further demonstrated that Egg is required intrinsically for controlling GSC self-renewal and proliferation. H3K9me3 but not H3K9me2 is eliminated in marked egg mutant GSCs. In addition, marked egg mutant GSCs are lost rapidly from the niche in comparison with the marked control GSCs, further supporting the idea that Egg is required for GSC maintenance. Moreover, the marked egg mutant GSCs and mitotic cysts are negative for TUNEL-based ApopTag labeling, but the marked 16-cell cysts in the regions 2b and 3 of the germarium are observed to be positive, indicating that Egg is dispensable for the survival of GSCs and early mitotic cysts but is required for the survival of 16-cell cysts. Finally, marked egg mutant GSCs appear to proliferate slower than the control GSCs based on cyst production and BrdU labeling. RNAi-mediated knockdown was used to show that loss of Egg function from GSCs and their progeny leads to the accumulation of DNA damage, suggesting that Egg is required for maintaining genome integrity. The accumulated DNA damage could also explain retarded GSC proliferation and increased 16-cell cyst apoptosis. These results demonstrate that Egg is required intrinsically for GSC self-renewal and proliferation and for the survival of 16-cell cysts (Wang, 2011).

BMP signaling and E-cadherin-mediated cell adhesion are essential for maintaining GSCs in the Drosophila ovary. BMP signaling represses bam-GFP expression and activates Dad-lacZ expression in GSCs. H3K9me3 is thought to be a histone marker for heterochromatin formation and transcriptional repression. Surprisingly, in marked egg mutant GSCs, bam-GFP remains repressed as in wild-type GSCs, but Dad-lacZ expression fails to be activated, indicating that Egg, and presumably H3K9me3, is dispensable for BMP signaling-mediated transcriptional repression of bam. The requirement of Egg for transcriptional activation of Dad could be indirect, but the detailed mechanism awaits further investigation. It was further demonstrated functionally that Egg controls GSC self-renewal by repressing a Bam/Bgcn-independent pathway by showing that marked bgcn egg double mutant GSCs are still lost at a much faster rate than marked control GSCs. Previously, Pumilio and Pelota were proposed to control GSC self-renewal by repressing a Bam/Bgcn-independent differentiation pathway as mutations for either factor can drive differentiation of bam mutant germ cells. Interestingly, mutations in egg can also cause differentiation of bgcn mutant germ cells, further supporting the idea that Egg represses a Bam/Bgcn-independent differentiation pathway to maintain GSC self-renewal. There are two possible strategies for Egg to repress differentiation and thus maintain GSC self-renewal: Egg represses the expression of a gene(s) important for GSC differentiation or activates the expression of a gene(s) critical for repressing GSC differentiation. Unfortunately, it remains unclear how Egg represses GSC differentiation to maintain self-renewal. Therefore, the identification of Egg target genes in GSCs will help define the unknown GSC differentiation pathway along with the identification of target genes of Pumilio and Pelota in order to gain a deeper understanding of GSC self-renewing mechanisms (Wang, 2011).

During the revision of this manuscript, a study was published to propose that Egg is required for H3K9me3 and heterochromatin formation in CBs and differentiated cysts, and is required for expression of piRNA genes and thus repression of transposable elements (TEs) (Rangan, 2011). Loss of piRNAs in germ cells is known to cause the activation of transposable elements (TEs) and consequently an increase in DNA damage. Consistently, this study shows that loss of egg function in germ cells leads to the accumulation of DNA damage. The regulation of piRNA by Egg offers mechanistic insight into why Egg is required for GSC maintenance and proliferation (Rangan, 2011). However, the current study has two different findings. One is that H3K9me3 establishment begins from GSCs, but not from CBs as the published study proposed. The other is that Egg is also required intrinsically for GSC maintenance and proliferation, but not for CB differentiation. The published study showed that spectrosome-containing single germ cells accumulate following germline-specific egg knockdown. In the current study, germline-specific expression of eggRNAi-1 leads to GSC loss, which is consistent with the mutant clonal analysis results, whereas the expression of eggRNAi-2 results in swollen germaria containing a few more spectrosome-containing CBs and cysts than control. The accumulation of the few more single germ cells is likely due to DNA damage-caused slowdown of mitotic progression. The difference between the published study and the current study could be simply caused by different egg knockdown efficiencies (Wang, 2011).

egg homozygous ovaries accumulate more undifferentiated germ cells and gradually lose their GSCs, which appear to be paradoxical. The egg mutant GSC loss phenotype can be attributed to the intrinsic requirement for GSC self-renewal. Further genetic analysis has revealed the requirement of Egg in ECs for controlling GSC differentiation by EC-specific RNAi-mediated egg knockdown. In the absence of Egg function from ECs, GSC progeny fail to differentiate and continuously proliferate as single germ cells, indicative of differentiation defects. In addition, loss of Egg function in ECs also causes EC loss, and in the complete absence of ECs, the progeny that have been generated before GSC loss also accumulate as single germ cells, further supporting that ECs are required for CB differentiation. Some of the accumulated single germ cells appear to upregulate Dad-lacZ expression and repress bam-GFP expression, suggesting that BMP signaling spreads to the germ cell differentiation niche, thereby interfering with germ cell differentiation. These findings suggest that Egg is required in ECs to promote germ cell differentiation at least in part by preventing self-renewal-promoting BMP signaling from spreading to the germ cell differentiation niche (Wang, 2011).

EFGR signaling has been suggested to act in ECs to control germ cell differentiation by repressing expression of Dally, a protein important for facilitating BMP diffusion. Interestingly, in the egg knockdown ECs, the expression of pERK, an EGFR signaling indicator, still remains normal, indicating that Egg is not essential for EGFR signaling in ECs. However, dally knockdown in ECs can partially suppress the egg knockdown mutant germ cell tumor phenotype, indicating that upregulation of dally in egg knockdown ECs contributes to BMP upregulation in the differentiation niche and to germ cell differentiation defects. The regulation of dally in ECs by Egg could be direct or indirect. The newly published study on Egg has shown that loss of Egg function in ECs leads to defective piRNA production and germ cell differentiation defects (Rangan, 2011). Consistently, this study also confirmed that egg knockdown in ECs results in dramatically increased expression of transposable elements (TEs). The germ cell differentiation defect can be rescued by a mutation in one of the DNA damage checkpoint genes, suggesting that DNA damage in ECs affects their ability to regulate germ cell differentiation. It will be of great interest to investigate if the mutation in the checkpoint gene also rescues defective BMP signaling in differentiated cells. Based on the findings from this study, it is proposed that Egg functions downstream of or in parallel with EGFR signaling to repress dally expression in ECs, thereby preventing BMP signaling from spreading to the differentiation niche. Because the signal(s) from ECs to control germ cell differentiation has not been identified yet, it remains unclear whether Egg also regulates additional factors independent of BMP signaling in ECs to control germ cell differentiation (Wang, 2011).

In this study, it was also shown that the egg knockdown ECs are gradually lost, and that GSCs cannot be maintained in the complete absence of ECs. This is consistent with the recently published finding that disruption of Rho function in ECs also cause EC loss and thus GSC loss (Kirilly, 2011). Because 5 to 6 most anteriorly localized ECs directly contact cap cells and GSCs, it is proposed that these ECs function as a part of the GSC niche to promote self-renewal by directly providing signals or indirectly by regulating cap cells function. A previous study suggests that JAK-STAT signaling functions in ECs to control GSC maintenance indirectly. How these GSC-contacting ECs contribute to GSC regulation remains to be further investigated (Wang, 2011).

piRNA production requires heterochromatin formation in Drosophila

Protecting the genome from transposable element (TE) mobilization is critical for germline development. In Drosophila, Piwi proteins and their bound small RNAs (piRNAs) provide a potent defense against TE activity. TE targeting piRNAs are processed from TE-dense heterochromatic loci termed ‘piRNA clusters’. While piRNA biogenesis from cluster precursors is beginning to be understood, little is known about piRNA cluster transcriptional regulation. This study shows that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. It is proposed that heterochromatin protects the germline by activating the piRNA pathway (Rangan, 2011).

The results indicate that dSETDB1 is required for transposon control in the germline and ovarian soma by positively regulating piRNA cluster transcription through the deposition of H3K9me3. To test directly whether transposon de-repression in either the germline or soma is sufficient to cause a block in GSC differentiation, two models of hybrid dysgenesis were used. First, the germline specific P-element model of hybrid dysgenesis was used, resulting in de-repression of P-element DNA transposons and progeny sterility when a male carrying a copy of an active P-element transposon (Harwich) is crossed to a female devoid of P-elements (w1118). This was attributed to the absence of maternally supplied piRNAs capable of silencing this transposon. Second, to test for somatic de-repression, flamenco mutant lines were used, which ablate production from the primary somatic piRNA cluster, flamenco, resulting in de-repression of gypsy-family transposable elements. In both cases germaria accumulate undifferentiated cells similar to those observed in dSETDB1 mutants. As with dSETDB1 mutants, these undifferentiated cells do not express Bam, but do stain positive for pMad. These results indicate that transposon mobilization alone in either germline or soma is sufficient to cause a block in GSC differentiation. Additionally, it was observed that the loss of GSC differentiation during P-element dysgenesis can be relieved by removing the Chk-2 kinase, suggesting that transposon de-repression activates a double stranded DNA break checkpoint. Interestingly, it is known that through viral-packaging, some gypsy-family elements maintain the ability to infect germline cells from the surrounding soma, leaving open the possibility that the GSC differentiation block in flamenco mutants is due to gypsy invasion and mobilization within differentiating germline cells. Alternatively, loss of piRNA production from the flamenco locus, as in piwi mutants, could result in the loss of somatic cells that surround germ cells and provide cues for differentiation. Thus transposon upregulation in the germline and soma in dSETDB1 mutants is sufficient to cause a loss of differentiation phenotype (Rangan, 2011).

Up regulation of transposons is sufficient to cause a block in differentiation From Drosophila to humans, a large fraction of eukaryotic genomes contain transposable elements. This study found a novel role for dSETDB1-mediated heterochromatin formation in activating transcription of piRNA clusters and thus triggering piRNA-based control of transposon regulations. Interestingly this transcriptional upregulation of the germline piRNA pathway happens at the time when transcription is generally upregulated in the germline to permit differentiations, potentially also leading to increases in transposon transcription. Thus production of piRNAs needed to keep transposon activity in check is timed to occur when they are likely most needed to protect the integrity of the genome of the next generation (Rangan, 2011).

Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling

Epigenetic gene regulation is essential for developmental processes. Eggless (Egg), the Drosophila orthologue of the mammalian histone methyltransferase, SETDB1, is known to be involved in the survival and differentiation of germline stem cells and piRNA cluster transcription during Drosophila oogenesis; however the detailed mechanisms remain to be determined. Using high-throughput RNA sequencing this study investigated target genes regulated by Egg in an unbiased manner. Egg was shown to play diverse roles in particular piRNA pathway gene expression, some long non-coding RNA expression, apoptosis-related gene regulation, and Decapentaplegic (Dpp) signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, this study demonstrate that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptotic cell death through activation of two pro-apoptotic genes, reaper and head involution defective. A model is proposed in which Egg regulates germ cell differentiation and apoptosis through canonical and noncanonical Dpp pathways in Drosophila oogenesis (Kang, 2018).

This study used RNA-seq data analysis and qRT-PCR validation to demonstrate that Egg plays diverse roles in the regulation of piRNA production, lncRNA expression, apoptosis-related gene expression, and Dpp signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, it was demonstrated that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptosis in vivo (Kang, 2018).

Regarding piRNA production, the results revealed that among the known piRNA machinery components, ago3, krimp, mael, and zuc genes are the major piRNA-related targets of Egg. Consistent with the previously suggested role of Egg for promoting piRNA production, all of the putative target genes were downregulated in egg mutant ovaries. In Drosophila, two piRNA processing pathways, primary processing and secondary processing, have been proposed; the primary processing pathway functions in both germline and somatic cells by processing precursor piRNAs into piRNAs whereas the secondary processing pathway functions only in germline cells in which piRNAs are amplified by the ping-pong cycle. Given the involvement of Egg in both germline and somatic piRNA production, tests were performed to see whether decreased expression of zuc is responsible for the ovarian phenotypes caused by loss of Egg. Phenotypic changes were investigated after introduction of wild-type zuc transgenes under the control of the actin5C-Gal4 driver into egg mutant background, but the ovaries of the genotypes (egg²¹³⁸/Df(2R)Dll-Mp; HA-tagged zuc (or EGFP-tagged zuc)/actin5C-Gal4) did not show any significant phenotypic changes compared with those of egg mutants. Ago3 is essentially involved in the secondary processing pathway along with Aub. The nuage, which surrounds the nuclei of nurse cells, has been proposed as a site for the ping-pong cycle. Various types of proteins, including Ago3, Krimp, and Mael, have been identified as nuage components. The decreased expression levels of the particular nuage components in egg mutant ovaries suggest that the reduction of germline piRNAs in egg mutants may result from not only a reduction of precursor piRNA transcription, as proposed previously, but also from a failure of the piRNA amplification pathway (Kang, 2018).

The data also revealed a previously unknown role of Egg as an lncRNA regulator. In Drosophila, the existence of lncRNAs has long been known, but only a few lncRNAs have been investigated. This study revealed that Egg is involved in regulating the expression of 100 potential lncRNA genes, and appears to play a repressive role in the expression of these lncRNAs. Among the upregulated lncRNAs in egg mutant ovaries, two well-known heat-inducible lncRNAs, αγ-element and hsr-ω are located in genomic regions where numerous TEs are found. Given the involvement of Egg in regulating piRNA production and thus TE mobilization, this raises an intriguing possibility of a link between the upregulation of αγ-element and hsr-ω in egg mutant ovaries and their genomic locations as TE hotspots. A strong upregulation of pncr003:2L and pncr004:X by loss of Egg is noteworthy, but the functional significance of these lncRNAs in the Drosophila ovary has not been determined. Although attempts were made to knock down pncr003:2L and pncr004:X using transgenic flies containing dsRNA for RNAi of pncr003:2L or pncr004:X under the control of the act5C-Gal4 driver, no phenotypic changes during oogenesis were detected. Interestingly, pncr003:2L was originally annotated as an lncRNA, but it was recently reported to encode two small functional peptides that are involved in regulating calcium transport in the Drosophila heart. Determination of the function of pncr003:2L in the Drosophila ovary is an interesting issue that needs to be addressed (Kang, 2018).

This study has demonstrated that dpp and dally were strongly upregulated in egg mutant ovaries and that dpp knockdown in ECs and early follicle cells in egg mutant background resulted in an increase in the size of the ovaries. Previously, by analysis of egg-RNAi knockdown in ECs, an EC-specific requirement for egg in controlling germ cell differentiation was suggested. Moreover, germ cell differentiation defects caused by egg knockdown in ECs was attributed to an increase in Dpp signaling because removal of one copy of dpp partially suppressed the tumorous phenotype caused by egg knockdown in ECs. The expression level of dally, an enhancer of Dpp signaling, may be maintained at a high level in the dpp knockdown in egg mutant background; therefore, the enhancement of Dpp signaling caused by loss of Egg may be maintained in the dpp knockdown in egg mutant germarium, thereby exhibiting similar differentiation defects as those observed in the egg mutant germarium. However, the defective cells in the dpp-knockdown in egg mutant background could be maintained for a longer time, which may be attributed to a reduction of the enhanced apoptosis in egg mutant ovaries because the increased expression levels of rpr and hid in egg mutant ovaries were significantly reduced in the dpp-knockdown in egg mutant background. Given that Egg is broadly expressed in germ cells and somatic cells in the ovary during oogenesis, dpp knockdown only in ECs and early follicle cells may not be sufficient to consistently counteract the overall apoptosis-promoting effect caused by dpp upregulation in egg mutant ovaries, which may explain the relatively low occurrence of the effect of the dpp knockdown in ECs and early follicle cells on the increase in ovary size (Kang, 2018).

A model is proposed in which loss of Egg may initially cause a relatively mild level of ectopic dpp and dally overexpression that may be sufficient to cause germ cell differentiation defects through repression of bam. Apoptosis may then be initiated when the level of ectopic dpp overexpression reaches a certain threshold level capable of inducing rpr and hid upregulation perhaps through a dpp positive-feedback loop. Alternatively, it cannot be ruled out that Egg represses dpp, dally, rpr and hid separately although the possibilities are not necessarily exclusive. Further studies are needed to determine the exact molecular mechanism by which Dpp signaling is increased in egg mutant ovaries. Given the role of BMPs in many mammalian stem cell systems and the existence of mammalian homologues of Egg and Dpp, the role of Egg in regulating Dpp signaling may provide important insights into their potential roles in mammalian stem cells (Kang, 2018).

The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells

The preservation of germ cell sexual identity is essential for gametogenesis. This study shows that H3K9me3-mediated gene silencing is integral to female fate maintenance in Drosophila germ cells. Germ cell specific loss of the H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, WDE, and HP1a, leads to ectopic expression of genes, many of which are normally expressed in testis. SETDB1 controls the accumulation of H3K9me3 over a subset of these genes without spreading into neighboring loci. At phf7, a regulator of male germ cell sexual fate, the H3K9me3 peak falls over the silenced testis-specific transcription start site. Furthermore, H3K9me3 recruitment to phf7 and repression of testis-specific transcription is dependent on the female sex determination gene Sxl. Thus, female identity is secured by an H3K9me3 epigenetic pathway in which Sxl is the upstream female-specific regulator, SETDB1 is the required chromatin writer, and phf7 is one of the critical SETDB1 target genes (Smolko, 2018).

In metazoans, germ cell development begins early in embryogenesis when the primordial germ cells are specified as distinct from somatic cells. Specified primordial germ cells then migrate into the embryonic gonad, where they begin to exhibit sex-specific division rates and gene expression programs, ultimately leading to meiosis and differentiation into either eggs or sperm. Defects in sex-specific programming interferes with germ cell differentiation leading to infertility and germ cell tumors. Successful reproduction, therefore, depends on the capacity of germ cells to maintain their sexual identity in the form of sex-specific regulation of gene expression (Smolko, 2018).

In Drosophila melanogaster, germ cell sexual identity is specified in embryogenesis by the sex of the developing somatic gonad. However, extrinsic control is lost after embryogenesis and sexual identity is preserved by a cell-intrinsic mechanism. The Sex-lethal (Sxl) female-specific RNA binding protein is an integral component of the cell-intrinsic mechanism, as loss of Sxl specifically in germ cells leads to a global upregulation of spermatogenesis genes and a germ cell tumor phenotype. Remarkably, sex-inappropriate transcription of a single gene, PHD finger protein 7 (phf7), a key regulator of male identity, is largely responsible for the tumor phenotype. Depletion of phf7 in mutants lacking germline Sxl suppresses the tumor phenotype and restores oogenesis. Moreover, forcing PHF7 protein expression in ovarian germ cells is sufficient to disrupt female fate and give rise to a germ cell tumor. Interestingly, sex-specific regulation of phf7 is achieved by a mechanism that relies primarily on alternative promoter choice and transcription start site (TSS) selection. Sex-specific transcription produces mRNA isoforms with different 5' untranslated regions that affect translation efficiency, such that PHF7 protein is only detectable in the male germline. Although the Sxl protein is known to control expression post-transcriptionally in other contexts the observation that germ cells lacking Sxl protein show defects in phf7 transcription argues that Sxl is likely to indirectly control phf7 promoter choice. Thus, how this sex-specific gene expression program is stably maintained remains to be determined (Smolko, 2018).

This study reports the discovery that female germ cell fate is maintained by an epigenetic regulatory pathway in which SETDB1 (aka EGGLESS, KMT1E, and ESET) is the required chromatin writer and phf7 is one of the critical SETDB1 target genes. SETDB1 trimethylates H3K9 (H3K9me3), a feature of heterochromatin. Using tissue-specific knockdown approaches this study established that germ cell specific loss of SETDB1, its protein partner WINDEI [WDE, aka ATF7IP, MCAF1 and hAM10], and the H3K9me3 reader, HP1a, encoded by the Su(var)205 locus, leads to ectopic expression of euchromatic protein-encoding genes, many of which are normally expressed only in testis. It was further found that H3K9me3 repressive marks accumulate in a SETDB1 dependent manner at 21 of these ectopically expressed genes, including phf7. Remarkably, SETDB1 dependent H3K9me3 deposition is highly localized and does not spread into neighboring loci. Regional deposition is especially striking at the phf7 locus, where H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific TSS. Lastly, this study found that H3K9me3 accumulation at many of these genes, including phf7, is dependent on Sxl. Collectively these findings support a model in which female fate is preserved by deposition of H3K9me3 repressive marks on key spermatogenesis genes (Smolko, 2018).

This study reveals a role for H3K9me3 chromatin, operationally defined as facultative heterochromatin, in securing female identity by silencing lineage-inappropriate transcription. H3K9me3 pathway members, the H3K9 methyltransferase SETDB1, its binding partner WDE, and the H3K9 binding protein HP1a, are required for silencing testis gene transcription in female germ cells. These studies further suggest a mechanism in which SETDB1, in conjunction with the female fate determinant Sxl, controls transcription through deposition of highly localized H3K9me3 islands on a select subset of these genes. The male germ cell sexual identity gene phf7 is one of the key downstream SETDB1 target genes. H3K9me3 deposition on the region surrounding the testis-specific TSS guaranties that no PHF7 protein is produced in female germ cells. In this model, failure to establish silencing leads to ectopic PHF7 protein expression, which in turn drives aberrant expression of testis genes and a tumor phenotype (Smolko, 2018).

Prior studies have established a role for SETDB1 in germline Piwi-interacting small RNA (piRNA) biogenesis and TE silencing. However, piRNAs are unlikely to contribute to sexual identity maintenance as mutations that specifically interfere with piRNA production, such as rhino, do not cause defects in germ cell differentiation. These findings, together with the observation that rhino does not control sex-specific phf7 transcription, suggests that the means by which SETDB1 methylates chromatin at testis genes is likely to be mechanistically different from what has been described for piRNA-guided H3K9me3 deposition on TEs.

The accumulation of H3K9me3 at many of these genes, including phf7, is dependent on the presence of Sxl protein. Thus, these studies suggest that Sxl is required for female-specific SETDB1 function. Sxl encodes an RNA binding protein known to regulate its target genes at the posttranscriptional levels. Sxl control may therefore be indirect. However, studies in mammalian cells suggest that proteins with RNA binding motifs are important for H3K9me3 repression, raising the tantalizing possibility that Sxl might play a more direct role in governing testis gene silencing. Further studies will be necessary to clarify how the sex determination pathway feeds into the heterochromatin pathway (Smolko, 2018).

phf7 stands out among the cohort of genes regulated by facultative heterochromatin because of its pivotal role in controlling germ cell sexual identity. Because ectopic protein expression is sufficient to disrupt female fate, tight control of phf7 expression is essential. phf7 regulation is complex, employing a mechanism that includes alternative promoter usage and TSS selection. This study reports that SETDB1/H3K9me3 plays a critical role in controlling phf7 transcription. In female germ cells, H3K9me3 accumulation is restricted to the region surrounding the silent testis-specific transcription start site. Dissolution of the H3K9me3 marks via loss of Sxl or SETDB1 protein is correlated with transcription from the upstream testis-specific site and ectopic protein expression, demonstrating the functional importance of this histone modification. Together, these studies suggest that maintaining the testis phf7 promoter region in an inaccessible state is integral to securing female germ cell fate (Smolko, 2018).

Although the loss of H3K9me3 pathway members in female germ cells leads to the ectopic, lineage-inappropriate transcription of hundreds of genes, integrative analysis identified only 21 SETDB1/H3K9me3 regulated genes. Given that one of these genes is phf7 and that ectopic PHF7 is sufficient to destabilize female fate, it is likely that inappropriate activation of a substantial number of testis genes is a direct consequence of ectopic PHF7 protein expression. How PHF7 is able to promote testis gene transcription is not yet clear. PHF7 is a PHD-finger protein that preferentially binds to H3K4me2, a mark associated with poised, but inactive genes and linked to epigenetic memory. Thus, one simple model is that ectopic PHF7 binds to H3K4me2 marked testis genes to tag them for transcriptional activation (Smolko, 2018).

It will be interesting to explore whether any of the other 20 SETDB1/H3K9me3 regulated genes also have reprogramming activity. In fact, ectopic fate-changing activity has already been described for the homeobox transcription factor Lim1 in the eye-antenna imaginal disc. However, whether Lim1 has a similar function in germ cells is not yet known. Intriguingly, protein prediction programs identify three of the uncharacterized testis-specific genes as E3 ligases. SkpE is a member of the SKP1 gene family, which are components of the Skp1-Cullin-F-box type ubiquitin ligase. CG12477 is a RING finger domain protein, most of which are believed to have ubiquitin E3 ligases activity. CG42299 is closely related to the human small ubiquitin-like modifier (SUMO) E3 ligase NSMCE2. Given studies that have linked E3 ligases to the regulation of chromatin remodeling, it is tempting to speculate that ectopic expression of one or more of these E3 ligases will be sufficient to alter cell fate. Future studies focused on this diverse group of SETDB1/H3K9me3 regulated genes and their role in reprogramming may reveal the multiple layers of regulation required to secure cell fate (Smolko, 2018).

The SETDB1-mediated mechanism for maintaining sexual identity uncovered in this study may not be restricted to germ cells. Recent studies have established that the preservation of sexual identity is essential in the adult somatic gut and gonadal cells for tissue homeostasis. Furthermore, the finding that loss of HP1a in adult neurons leads to masculinization of the neural circuitry and male specific behaviors suggests a connection between female identity maintenance and H3K9me3 chromatin. Thus, it is speculated that SETDB1 is more broadly involved in maintaining female identity (Smolko, 2018).

These studies highlight an emerging role for H3K9me3 chromatin in cell fate maintenance. In the fission yeast S. pombe, discrete facultative heterochromatin islands assemble at meiotic genes that are maintained in a silent state during vegetative growth. Although less well understood, examples in mammalian cells indicate a role for SETDB1 in lineage-specific gene silencing. Thus, silencing by SETDB1 controlled H3K9 methylation may be a widespread strategy for securing cell fate. Interestingly, H3K9me3 chromatin impedes the reprogramming of somatic cells into pluripotent stem cells (iPSCs). Conversion efficiency is improved by depletion of SETDB1. If erasure of H3K9me3 via depletion of SETDB1 alters the sexually dimorphic gene expression profile in reprogrammed cells, as it does in Drosophila germ cells, the resulting gene expression differences may cause stem cell dysfunction, limiting their therapeutic utility (Smolko, 2018).

Su(var)2-10 and the SUMO pathway link piRNA-guided target recognition to chromatin silencing

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. This study shows that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1 (Eggless)/Wde histone methyltransferase effector. It is proposed that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites (Ninova, 2020a).

The majority of transcriptional control is achieved by transcription factors that bind short sequence motifs on DNA. In many eukaryotic organisms, transcriptional repression can also be guided by small RNAs, which (in complex with Argonaute proteins) recognize their genomic targets using complementary interactions with nascent RNA. Small RNA-based regulation provides flexibility in target selection without the need for new transcription factors and as such is well suited for genome surveillance systems to identify and repress the activity of harmful genetic elements such as transposons (Ninova, 2020a).

Transcriptional repression guided by small RNAs correlates with the deposition of repressive chromatin marks, particularly histone 3 lysine 9 methylation (H3K9me) in S. pombe, plants, and animals. In addition, plants and mammals also employ CpG DNA methylation for target silencing. Small RNA/Ago-induced transcriptional gene silencing is best understood in S. pombe, where the RNA-induced transcriptional silencing complex (RITS) was studied biochemically and genetically. In contrast to yeast, the molecular mechanism of RITS in Metazoans remains poorly understood. Small RNA-induced transcriptional repression mechanisms might have independently evolved several times during evolution and thus might mechanistically differ from that of S. pombe (Ninova, 2020a).

In Metazoans, small RNA-guided transcriptional repression is mediated by Piwi proteins, a distinct clade of the Argonaute family, and their associated Piwi-interacting RNAs (piRNAs). Both in Drosophila and in mouse, the two best-studied Metazoan systems, nuclear Piwis are responsible for transcriptional silencing of transposons. Based on the current model, targets are recognized through binding of the Piwi/piRNA complex to nascent transcripts of target genes. In both Drosophila and mouse, piRNA-dependent silencing of transposons correlates with accumulation of repressive chromatin marks (H3K9me3 and, in mouse, CpG methylation of DNA) on target sequences. These marks can recruit repressor proteins, such as HP1, providing a mechanism for transcriptional silencing. However, how recognition of nascent RNA by the Piwi/piRNA complex leads to deposition of repressive marks at the target locus is not well understood. Several proteins, Asterix (Arx)/Gtsf1, Panoramix (Panx)/Silencio, and Nxf2, were shown to associate with Piwi and are required for transcriptional silencing. Accumulation of H3K9me3 on Piwi/Panx targets requires the activity of the histone methyltransferase SetDB1 (also known as Egg). However, a mechanistic link between the Piwi/Arx/Panx/Nxf2 complex, which recognizes targets, and the effector chromatin modifier has not been established (Ninova, 2020a and references therein).

This study identified Su(var)2-10/dPIAS to provide the link between the Piwi/piRNA and the SetDB1 complex in piRNA-induced transcriptional silencing. In Drosophila, Su(var)2-10 mutation causes suppression of position effect variegation, a phenotype indicative of its involvement in chromatin repression. Su(var)2-10 associates with chromatin and regulates chromosome structure. It also emerged in screens as a putative interactor of the central heterochromatin component HP1, a repressor of enhancer function, and a small ubiquitin-like modifier (SUMO) pathway component. However, its molecular functions in chromatin silencing were not investigated. Su(var)2-10 belongs to the conserved PIAS/Siz protein family, of which the yeast, plant, and mammalian homologs act as E3 ligases for SUMOylation of several substrates. This paper reports the role of Su(var)2-10 in germ cells of the ovary, where chromatin maintenance and transposon repression are essential to grant genomic stability across generations. Germ cell depletion of Su(var)2-10 phenocopies loss of Piwi; both lead to strong transcriptional activation of transposons and loss of repressive chromatin marks over transposon sequences. Su(var)2-10 genetically and physically interacts with Piwi and its auxiliary factors, Arx and Panx. It was demonstrated that the repressive function of Su(var)2-10 is dependent on its SUMO E3 ligase activity and the SUMO pathway. These data point to a model in which Su(var)2-10 acts downstream of the piRNA/Piwi complex to induce local SUMOylation, which in turn leads to the recruitment of the SetDB1/Wde complex. SUMO modification was shown to play a role in the formation of silencing chromatin in various systems from yeast to mammals, including the recruitment of the silencing effector SETDB1 and its co-factor MCAF1 by repressive transcription factors. Together, these findings indicate that the piRNA pathway utilizes a conserved mechanism of silencing complex recruitment through SUMOylation to confer transcriptional repression (Ninova, 2020a).

In both insect and mammals, piRNA-guided transcriptional silencing is associated with the deposition of repressive chromatin marks on genomic targets. In Drosophila, the conserved histone methyltransferase SetDB1 (Egg) is responsible for deposition of the silencing H3K9me3 mark at Piwi targets. However, the molecular mechanism leading to the recruitment of SetDB1 by the Piwi/piRNA complex remained unknown. Thus study showed that in Drosophila SUMO and the SUMO E3 ligase Su(var)2-10 act together downstream of the piRNA-guided complex to recruit the histone methyltransferase complex SetDB1/Wde and cause transcriptional silencing. The results suggest a model for the molecular mechanism of piRNA-guided transcriptional silencing in which Su(var)2-10 provides the connection between the target recognition complex composed of piRNA/Piwi/Panx/Arx and the chromatin effector complex composed of SetDB1 and Wde (Ninova, 2020a).

This study has identified a new role for the SUMO pathway in piRNA-guided transcriptional silencing. The SUMO pathway plays important roles in heterochromatin formation and maintenance, and genome stability in different organisms from yeast to humans. Among different functions, SUMO is required for recruitment and activity of the histone methyltransferase complex composed of SetDB1 and MCAF1 (Wde in Drosophila), which confers transposon silencing in mammals. Remarkably, SUMO-dependent recruitment of SetDB1 to TEs in mammalian somatic cells does not require piRNAs but is instead mediated by the large vertebrate-specific family of Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs) that bind specific DNA motifs. Although distinct members of the KRAB-ZFP family recognize different sequence motifs in target transposons, repression of all targets by various KRAB-ZFPs requires the universal co-repressor KAP1/TIF1b (KRAB-associated protein 1). KAP1 is a SUMO E3 ligase, and its auto-SUMOylation leads to SetDB1 recruitment. The current results suggest that Drosophila Su(var)2-10 can be SUMOylated, and SetDB1 and Wde have functional SIMs, suggesting that Su(var)2-10 auto-SUMOylation might induce SetDB1/Wde recruitment. These results suggest that two distinct transposon repression pathways, by DNA-binding proteins and by piRNAs, both rely on SUMO-dependent recruitment of the conserved silencing effector to the target (Ninova, 2020a).

The results in Drosophila and studies in mammals suggest that in both clades self-SUMOylation of SUMO E3 ligases might be involved in recruitment of SetDB1 to chromatin. However, these results do not exclude the possibility that the recruitment of SetDB1 is facilitated by SUMOylation of additional chromatin proteins by Su(var)2-10. Studies in yeast led to the 'SUMO spray' hypothesis that postulates that SUMOylation of multiple different proteins localized in physical proximity promotes the assembly of multi-unit effector complexes. Local concentration of multiple SUMO moieties leads to efficient recruitment of SUMO-interacting proteins. According to this hypothesis, multiple SUMO-SIM interactions within a protein complex act synergistically, and thus SUMOylation of any single protein is neither necessary nor sufficient to trigger downstream processes. Assembly of such 'SUMO spray' on chromatin might be governed by the same principles of multiple weak interactions as was recently recognized for the formation of various phase-separated liquid-droplet compartments in the cell. The presence of Su(var)2-10 on a chromatin locus might lead to SUMOylation of multiple chromatin-associated proteins that are collectively required for the recruitment of effector chromatin modifiers. The SUMOylation consensus (ΨKxE/D) is very simple and therefore quite common in the fly proteome. Consistent with this, several hundred SUMOylated proteins were identified in proteomic studies in Drosophila. Thus, it is possible that collective SUMOylation of multiple chromatin-associated proteins contributes to recruitment and stabilization of the SetDB1 complex on chromatin (Ninova, 2020a).

The cascade of events leading to repression initiated by target recognition by piRNA/Piwi, followed by interaction with Su(var)2-10 and subsequent SUMO-dependent recruitment of SetDB1/Wde, suggests that the three complexes tightly cooperate. But do these three complexes (Piwi, Su(var)2-10, and SetDB1) always work together, or does each complex have additional functions independent of the other two? Genome-wide analysis suggests that the vast majority of Piwi targets are repressed through SUMO/Su(var)2-10 and, likely, SetDB1/Wde, suggesting that Piwi always requires these other complexes for its function in transcriptional silencing. On the other hand, multiple instances were found of host genes that are repressed by Su(var)2-10 and SetDB1 but do not require piRNAs. Su(var)2-10 and SetDB1 are also expressed outside of the gonads and were implicated in chromatin silencing in somatic tissues that lack an active piRNA pathway. It is speculated that Su(var)2-10 might bind to specific targets directly through its SAP domain or might get recruited by specific DNA-binding proteins, similar to the way SetDB1 is recruited to ERVs by KRAB-ZFP in mammals, though specific factors are yet to be uncovered (Ninova, 2020a).

Though both Drosophila and mouse have nuclear Piwi proteins involved in transcriptional silencing of transposons, these proteins, PIWI and MIWI2, are not one-to-one orthologs. Unlike Drosophila, other insects including the silkworm Bombyx mori, the flour beetle Tribolium castaneum, and the honeybee Apis mellifera encode only two Piwi proteins, and at least in B. mori, these proteins do not localize to the nucleus. These observations suggest that the nuclear Piwi pathway in Drosophila has evolved independently in this lineage. In light of this evolutionary interpretation, the interaction of the Piwi complex and the E3 SUMO ligase Su(var)2-10 indicates that in Drosophila the nuclear piRNA pathway co-opted an ancient mechanism of SUMO-dependent recruitment of the histone-modifying complex for transcriptional silencing of transposons. The molecular mechanism of piRNA-induced transcriptional repression in other clades such as mammals might have evolved independently of the corresponding pathway in flies. It will be interesting to investigate if mammals also use SUMO-dependent recruitment of silencing complexes for transcriptional repression of piRNA targets (Ninova, 2020a).

The SUMO ligase Su(var)2-10 controls hetero- and euchromatic gene expression via establishing H3K9 trimethylation and negative feedback regulation

Chromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. This study found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1 (Eggless)/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis (Ninova, 2020b).

This study shows that in addition to the effects on TE silencing (Ninova, 2020a), Su(var)2-10 and H3K9me3 influence the expression of protein-coding genes. Su(var)2-10-dependent H3K9me3 deposition on TEs affects the expression of genes located in heterochromatin and of euchromatic genes adjacent to TE insertions. Su(var)2-10 is also involved in TE-independent H3K9me3 deposition on host genes, which is essential for the suppression of ectopic expression of tissue-specific genes, thereby conferring correct cell type identity (Ninova, 2020b).

Approximately half of the human genome comprises TE sequences, and the TE fraction is as high as 90% in several plant species. One new TE insertion per generation is estimated to propagate to the offspring. Somatic TE insertions, although difficult to detect, are likely even more prevalent. Thus, TE activity is a major source of genetic variation that can occur on a very short timescale. The effects of TEs on the host transcriptome have been the subject of many studies ever since Barbara McClintock identified 'control' elements that regulate gene expression before genome compositions were known. TEs can disrupt gene expression by inserting into coding regions or into or close to cis-regulatory sequences. TE insertions are not always disruptive: insertions into non-coding regions can bring new regulatory elements that change gene expression patterns, resulting in increased fitness. Instances of positive selection for TE insertions are well documented in Drosophila. TE-derived promoters also drive the expression of numerous mouse and human genes, suggesting that TE insertions can be co-opted into gene regulatory pathways (Ninova, 2020b).

In addition to changes in the DNA sequence, TE insertions may introduce local epigenetic effects. Active TEs are transcriptionally silenced by H3K9 trimethylation and/or DNA methylation. The H3K9me3 mark can spread several kilobases outside the TE region, affecting adjacent cis-regulatory elements of host genes, and thereby interfering with their normal expression. TE insertions with high levels of H3K9me3 are strongly selected against, supporting a model that TEs can alter the expression of host genes through epigenetic changes (Ninova, 2020b).

The finding that Su(var)2-10 is responsible for the deposition of H3K9me3 on TE bodies and flanking sequences allows separation if the effect of direct damage to cis-regulatory elements from the effect on chromatin. Evidence was found that TE insertions can lead to H3K9me3-dependent changes in gene expression, as shown for the jheh3 and frl loci. Notably, the BARI insertion at the jheh3 locus was shown to be positively selected in the D. melanogaster population, indicating that Su(var)2-10-dependent epigenetic silencing caused by a TE insertion can be used for beneficial rewiring of host gene regulatory networks (Ninova, 2020b).

The current results suggest that TEs can rewire gene regulatory networks on a short timescale, at least in part via their effects on chromatin. Euchromatic H3K9me3 peaks due to TE insertions are widespread in Drosophila, indicating that TE insertions may be a common cause of gene regulatory variation. New TE insertions during development generate genomic diversity between different cell types in human and mouse with implications for tumorigenesis and brain development. Future studies are required to elicit the epigenetic effects of somatic TE insertions on gene regulatory networks (Ninova, 2020b).

Heterochromatin domains include nearly 30% of the fly genome. Although relatively gene-poor, heterochromatin hosts several hundred protein-coding genes. Studies of chromosomal rearrangements suggested that heterochromatic localization is required for the proper expression of heterochromatic genes. However, the molecular mechanism of the positive effect of the heterochromatin environment on expression is not fully understood (Ninova, 2020b).

Consistent with previous studies, this study observed many active genes in H3K9me3-rich heterochromatic regions and found that for many active heterochromatic genes, Su(var)2-10-induced H3K9 methylation is not only permissive but also required for proper expression (Ninova, 2020b).

How can the same chromatin mark lead to the repression of genes in euchromatin and activation in heterochromatin? H3K9me3 is present over the gene bodies and regions flanking heterochromatic genes, but is depleted at promoters, which instead carry typical active marks such as H3K4me3 and Pol II occupancy. Thus, H3K9me3 over gene bodies appears to be compatible with transcription. H3K9me3 loss upon Su(var)2-10 GLKD correlated with increased levels of intronic RNAs and the appearance of H3K4me2/3 and Pol II signals in introns, indicating the upregulation of spurious transcripts originating from within host-gene introns. One possible source of such transcripts is the activation of TE promoters that are highly abundant within introns and flanking sequences of heterochromatic genes. It is proposed that transcription from TE promoters located in introns and flanking sequences interferes with proper gene expression through transcriptional interference (Ninova, 2020b).

H3K9me3 loss also disrupted the normal isoform regulation of heterochromatic genes, as was observed both truncated and extended mRNA isoforms with coding potential distinct from the canonical gene mRNA upon the depletion of Su(var)2-10. The activation of cryptic promoters may disrupt proper gene expression through multiple mechanisms, such as reduction in canonical mRNA output or dominant negative effects of the extended or truncated protein isoforms. Not all heterochromatic genes that lose H3K9me3 upon Su(var)2-10 germline knockdown (GLKD) show signs of interfering transcripts or cryptic promoters, indicating that H3K9me3 may have other functions in heterochromatic gene activation. For example, the compaction of heterochromatin by HP1 may bring distant enhancers of heterochromatic genes into physical proximity of promoters to activate expression. The results, combined with previous studies, indicate that genes positioned in heterochromatin require high H3K9me3 levels for proper expression and isoform selection (Ninova, 2020b).

Discrete Su(var)2-10-dependent H3K9me3 peaks are present in a number of euchromatic genes. Some of these peaks have no TEs in their vicinity, and their H3K9me3-based repression is conserved between D. melanogaster and D. virilis, two species that separated >45 million years ago and have no common TE insertions. The expression of many of these TE-independently repressed genes is restricted to specific tissues such as testis, the digestive system, or the CNS, and the loss of H3K9me3 leads to ectopic expression in the female germline. The finding is in line with a recent report that SetDB1 depletion in the female germline was associated with the loss of H3K9me3 and the mis-expression of male-specific genes. H3K9me3, SetDB1, and the SUMO pathway were also implicated in lineage-specific gene expression and cell fate commitment in mammals. These data suggest that a TE-independent H3K9me3 deposition via the SUMO-SetDB1 pathway plays an evolutionarily conserved role in restricting gene expression to proper cell lineages (Ninova, 2020b).

SUMO- and Su(var)2-10-dependent H3K9me3 repression also regulates several factors involved in heterochromatin formation and maintenance, such as SUMO (smt3), Wde, Sov, and CG30403. Wde is the homolog of the mammalian MCAF1/ATF7IP, which is required for the nuclear localization and stability of SetDB1 and promotes its methyltransferase activity. Drosophila Wde also associates with SetDB1, and their germline depletion results in a similar phenotype, supporting the role of Wde as a conserved SetDB1 co-factor. The current data in Drosophila and studies in mammals suggest that SUMO is involved in SetDB1/Wde recruitment to its targets. HP1 is an H3K9me3 reader that is responsible for the structural properties of heterochromatin and also serves as a hub for many other heterochromatin proteins. Both Sov and CG30403 interact with HP1, and Sov is critical for heterochromatin maintenance (Ninova, 2020b).

The genes encoding Wde, SUMO, Sov, and CG30403 reside in euchromatin and are repressed by local H3K9me3. Unlike tissue-restricted genes, which are often completely repressed by Su(var)2-10 in the female germline, these factors are not fully silenced, although they are upregulated upon Su(var)2-10 depletion. The results indicate that these four genes are part of a negative feedback mechanism that controls heterochromatin formation. Negative feedback in biological circuits maintains protein levels within a certain range, providing homeostatic regulation. It is proposed that SUMO-dependent repression of heterochromatin proteins provides such homeostatic regulation to maintain the proper ratio and boundaries of hetero- and euchromatin. According to this model, specific genes, such as wde, act as sensors of the overall H3K9me3 level. Insufficient levels of H3K9 methylation lead to elevated sensor gene expression due to decreased H3K9me3 at their promoters, which in turn enhances H3K9me3 deposition and heterochromatin formation throughout the genome. Concomitant repression of sensor genes ensures that H3K9me3 is restricted to proper genomic domains and does not spread to euchromatic regions that should remain active. Inspection of ENCODE data showed that the mammalian homolog of wde, ATF7IP, is decorated by H3K9me3 in some human cell lines, suggesting that this mode of regulation may be deeply conserved (Ninova, 2020b).

A reminiscent negative feedback loop was identified in yeast. The single H3K9 methyltransferase clr4 is suppressed by H3K9me3 to restrict ectopic spreading of silencing chromatin. In mammals, genes encoding proteins from the KRAB-ZFP family of transcriptional repressors reside in H3K9me3- and HP1-enriched loci. Thus, autoregulation of heterochromatin effectors is a conserved mode of chromatin regulation, although the genes involved in the feedback mechanism differ between different organisms. In the future, it will be important to dissect the network architecture of heterochromatin regulation. As heterochromatin formation and maintenance was reported to be disrupted in cancer and during aging, this mechanism may be a promising target of therapeutic interventions (Ninova, 2020b).

H3K9me3 writer enzymes are targeted to genomic loci by different mechanisms. In the case of TE repression in germ cells, piRNAs bound to nuclear Piwi proteins serve as sequence-specific guides that bind complementary nascent transcripts and recruit Su(var)2-10, which induces H3K9me3 deposition by SetDB1. Su(var)2-10 identifies non-TE targets in a piRNA-independent fashion, in agreement with a broader function of Su(var)2-10 in development. The observation that H3K9me3 peaks at homologous euchromatic genes are also present in the distantly related D. virilis points to a conserved mechanism of H3K9me3 deposition in host-gene regulation (Ninova, 2020b).

The molecular mechanism of piRNA-independent recruitment of Su(var)2-10 remains to be explored. Su(var)2-10 has a putative DNA binding SAP domain that may be sufficient for its binding to DNA. However, motif enrichment analysis failed to identify a common sequence motif among TE-independent Su(var)2-10 targets (MEME-ChIP), suggesting that different partners may recruit Su(var)2-10 to distinct targets. In mammals, a large family of transcription factors, the KRAB-ZFPs, are responsible for SetDB1 recruitment and H3K9me3 deposition on many different targets, primarily endogenous retroviruses. Individual members of the KRAB-ZFP family influence distinct targets due to differences in DNA-binding specificities of their zinc-finger DNA-binding domains. Notably, SetDB1 recruitment through KRAB-ZFPs occurs through a SUMO-dependent mechanism. The KRAB-ZFP family is vertebrate specific, and there are no known proteins in D. melanogaster that can recruit H3K9me3 activity. A preliminary search for direct Su(var)2-10 interactors using a yeast two-hybrid screen identified several proteins with putative DNA-binding domains. Thus, it is proposed that analogous to the KRAB-ZFP pathway in mammals, Su(var)2-10 may link DNA-binding proteins to the SetDB1 silencing machinery. Future studies are necessary to identify the proteins that guide Su(var)2-10 to target loci and to elucidate TE-independent recruitment mechanisms of the silencing machinery (Ninova, 2020b).

Search PubMed for articles about Drosophila Eggless

Bilodeau, S., et al. (2009). SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. Genes Dev 23: 2484–2489. PubMed ID: 19884255

Clough, E., Moon, W., Wang, S., Smith, K. and Hazelrigg, T. (2007). Histone methylation is required for oogenesis in Drosophila. Development 134(1): 157-65. PubMed ID: 17164421

Dodge, J. E., Kang, Y. K., Beppu, H., Lei, H. and Li, E. (2004). Histone H3K9 methyltransferase ESET is essential for early development. Mol. Cell. Biol. 24: 2478-2486. PubMed ID: 14993285

Kang, I., Choi, Y., Jung, S., Lim, J. Y., Lee, D., Gupta, S., Moon, W. and Shin, C. (2018). Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling. Sci Rep 8(1): 7123. PubMed ID: 29740006

Kirilly, D., Wang, S. and Xie, T. (2011). Self-maintained escort cells form a germline stem cell differentiation niche. Development 138: 5087–5097. PubMed ID: 22031542

Koch, C. M., Honemann-Capito, M., Egger-Adam, D. and Wodarz, A. (2009). Windei, the Drosophila homolog of mAM/MCAF1, is an essential cofactor of the H3K9 methyl transferase dSETDB1/Eggless in germ line development. PLoS Genet 5: e1000644. PubMed ID: 19750210

Li, H., Rauch, T., Chen, Z.-X., Szabo, P., Riggs, A. D. and Pfeifer, G. (2006). The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells. J. Biol. Chem. 281: 19489-19500. PubMed ID: 16682412

Ninova, M., Chen, Y. A., Godneeva, B., Rogers, A. K., Luo, Y., Fejes Toth, K. and Aravin, A. A. (2020a). Su(var)2-10 and the SUMO pathway link piRNA-guided target recognition to chromatin silencing. Mol Cell 77(3): 556-570. PubMed ID: 31901446

Ninova, M., Godneeva, B., Chen, Y. A., Luo, Y., Prakash, S. J., Jankovics, F., Erdelyi, M., Aravin, A. A. and Fejes Toth, K. (2020b). The SUMO ligase Su(var)2-10 controls hetero- and euchromatic gene expression via establishing H3K9 trimethylation and negative feedback regulation. Mol Cell 77(3): 571-585 e574. PubMed ID: 31901448

Rangan P, Malone CD, Navarro C, Newbold SP, Hayes PS, et al. (2011) piRNA production requires heterochromatin formation in Drosophila. Curr. Biol. 21: 1373–1379. PubMed ID: 21820311

Sarraf, S. A. and Stancheva, I. (2004). Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol. Cell 15: 595-605. PubMed ID: 15327775

Schultz, D. C., Ayyanathan, K., Negorev, D., Maul, G. G. and Rauscher, F. J., III (2002). SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 16: 919-932. PubMed ID: 11959841

Seum, C., et al. (2007). Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet 3: e76. PubMed ID: 17500594

Smolko, A. E., Shapiro-Kulnane, L. and Salz, H. K. (2018). The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells. Nat Commun 9(1): 4155. PubMed ID: 30297796

Wang, H., An, W., Cao, R., Xia, L., Erdjument-Bromage, H., Chatton, B., Tempst, P., Roeder, R. G. and Zhang, Y. (2003). mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression. Mol. Cell 12: 475-487. PubMed ID: 14536086

Wang, X., et al. (2011). Histone H3K9 trimethylase Eggless controls germline stem cell maintenance and differentiation. PLoS Genet. 7(12): e1002426. PubMed ID: 22216012

Yang, L., Xiam, L., Wu, D. Y., Wang, H., Chansky, H. A., Schubach, W. H., Hickstein, D. D. and Zhang, Y. (2002). Molecular cloning of ESET, a novel histone H3-specific methyltransferase that interacts with ERG transcription factor. Oncogene 21: 148-152. PubMed ID: 11791185

Yang, L., Mei, Q., Zielinska-Kwiatkowska, A., Matsui, Y., Blackburn, M. L., Benedetti, D., Krumm, A. A., Taborsky, G. J., Jr and Chansky, H. A. (2003). An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B. Biochem. J. 369: 651-657. PubMed ID: 12398767

Yoon J, et al. (2008). dSETDB1 and SU(VAR)3–9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster. PLoS ONE 3: e2234. PubMed ID: 18493619

date revised: 15 April 2020

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