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).