InteractiveFly: GeneBrief

PHD finger protein 7 ortholog : Biological Overview | References

BIOLOGICAL OVERVIEW

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. This study identified PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). It is proposed that PHF7 acts as a conserved epigenetic 'reader' that activates the male germline sexual program (Yang, 2012).

Sex determination is key to sexual reproduction, and both somatic cells and germ cells need to establish sex-specific developmental fates. Germline sexual development is essential for the production of two distinct gametes, and underlies important differences in the regulation of male versus female fertility. In some species, germline stem cells are present in both males and females to sustain constant gamete production, but are regulated differently throughout development. In other species such as humans, sex-specific germ cell development produces a germline stem cell population only in males, whereas females have a much more limited capacity in making eggs. Defects in germline sexual development lead to a failure in gametogenesis, thus the study of germline sex determination is essential for understanding normal reproductive potential and treating infertility (Yang, 2012).

In some animals, such as mammals and Drosophila, the sex chromosome compositions of the soma and germline are interpreted independently, and the 'sex' of the germline must match that of the soma for proper germ cell development to occur. For example, patients with Klinefelter's Syndrome have an XXY sex chromosome constitution and are almost always infertile. These individuals develop somatically as males due to the presence of a Y chromosome but the germline suffers from severe atrophy, including the loss of premeiotic germline and germline stem cells. This is due to the presence of two X chromosomes in the germ cells, as the limited spermatogenesis in these patients is from germ cells that have lost one of the X chromosomes. In Drosophila, XX females that are somatically transformed into males exhibit a similar germline loss due to a conflict in sexual identity between the masculinized soma and XX germline. Thus, fruit flies are a valuable model organism for studying how germ cells establish a proper sexual identity by coordinating intrinsic signals and those coming from the soma (Yang, 2012).

In Drosophila, the presence of two X chromosomes promotes female somatic identity by activating an alternative splicing cascade that acts through Sex lethal (SXL) and Transformer (TRA), and ultimately leads to production of either the male or female forms of the transcription factors Doublesex (DSX) and Fruitless (FRU). DSX and FRU are responsible for virtually all sexually dimorphic somatic traits in Drosophila, with DSX being the key factor in the somatic gonad. In contrast, the germline does not determine its sex with this cascade and factors like TRA and DSX are not required in germ cells. Although SXL is required to promote female germ cell identity, its targets and mechanism of action in the germline are not known. The transcription factor OVO and the ubiquitin protease Ovarian Tumor (OTU) are also required in the female germline and thought to function upstream of SXL. Even less is known about how sexual identity is specified in male germ cells. Male germ cells receive a signal through the JAK/STAT pathway that promotes their sexual identity, but the downstream factors that are subsequently activated are not known. Similarly, how male germ cells respond to their own sex chromosome constitution is also not known (Yang, 2012).

This study reports a histone code reader, Plant Homeodomain (PHD) Finger 7 (PHF7), that acts in the Drosophila germline to promote male sexual identity. PHF7 is specifically expressed in male germ cells from early stages of development and is restricted to male germline stem cells (GSCs) and spermatogonia. Phf7 is required for GSC maintenance and proper entry into spermatogenesis. Interestingly, expression of Phf7 in female germ cells causes ablation of the female germline. Moreover, Phf7 affects sexual compatibility between germline and soma. Loss of Phf7 in XY germ cells alleviates the germline loss typically observed when XY germ cells are surrounded by a female soma, and expression of Phf7 can induce spermatogenesis in XX germ cells nurtured by male soma. These findings indicate that Phf7 is an essential factor in determining sexual development in the Drosophila germline, and suggest that activation of the male identity occurs through interaction with the germline epigenome (Yang, 2012).

The data indicate that Phf7 acts to promote a male identity in the germline. Loss of Phf7 function affected male GSC maintenance and spermatogenesis, but had no effect in females. Phf7-mutant GSCs exhibited a more female-like pattern of spectrosome localization, and male (XY) germ cells mutant for Phf7 were more compatible with a female soma than were wild-type male germ cells. Further, expression of PHF7 was able to masculinize the female germline: PHF7 expression induced apoptosis in developing XX germ cells and interacted with mutations in otu in a manner that indicates XX germ cells that express PHF7 are more male-like. Strikingly, PHF7 expression was able to induce spermatogenesis in XX germ cells when they are present in a male soma, something that XX germ cells are normally not able to do. Taken together, these results indicate that Phf7 promotes and is sufficient to induce male identity in the germline (Yang, 2012).

Sex determination is thought to be initiated early during development, and sex-specific differences in the male and female germline are first observed during embryogenesis. The data indicate that Phf7 plays a role in early germline sexual development, rather than a late role to regulate germ cell differentiation and gametogenesis. First, PHF7 expression is observed in the embryonic gonad and, in the adult, PHF7 is found in the GSCs and early gonia and disappears dramatically as gonia transition to spermatocytes. Further, forced PHF7 expression disrupts early female germ cell development, around the time when they are first forming GSCs. Expression of PHF7 after the early cystoblast stage (Bam-Gal4, UAS-Gal4) had no effect on the female germline, indicating that it can only affect early stages of female germ cell development. Phf7 mutants show defects in male GSC behavior and maintenance, and in the initial progression to form spermatocytes, but it is possible that these defects are due to even earlier problems in male sexual identity (Yang, 2012).

Germline sexual identity is determined by both the germ cell sex chromosome constitution and signals from the surrounding soma. Phf7 expression is activated in XX germ cells when in contact with a male soma and repressed in XY germ cells when contacting a female soma. However, in a female somatic environment, XY germ cells are somewhat more likely than XX germ cells to express Phf7, indicating that Phf7 may also respond to the sex chromosome constitution of the germ cells in addition to being regulated by the soma. Further, exogenous expression of Phf7 is required to promote spermatogenesis in XX germ cells when in a male soma. Thus, the Phf7 expression that is normally initiated in such germ cells by the male soma must either not be maintained, or may be insufficient to overcome the influence of the XX sex chromosome genotype (Yang, 2012).

It is likely that Phf7 is not acting alone to control male sexual identity. Phf7 mutant males are still able to undergo spermatogenesis, but at a much reduced capacity. This appears to be the null phenotype for Phf7 as ther mutants have lost significant portions of the coding sequence. Further, when PHF7 is expressed in XX germ cells present in a male soma, these germ cells can undergo spermatogenesis, but the penetrance of this phenotype is low. Interestingly, the rescue of spermatogenesis in these XX germ cells follows an 'all or nothing' pattern; either the rescue is largely complete to give full testes and sperm production, or little rescue is observed. Therefore, there appears to be a threshold that must be crossed to promote male germline sexual identity, and that once this threshold is met, those germ cells either take over the testis, or induce other germ cells to also follow the male pathway. The simplest explanation for both the incomplete block to spermatogenesis in Phf7 mutants and the incomplete rescue of spermatogenesis by Phf7 in XX males is that an additional factor (or factors) exists that promotes male identity in addition to Phf7. Such a factor could function parallel to Phf7 in a single pathway, or represent independent input regarding germline sex determination (e.g., independent signals from the soma that influence germline sex) (Yang, 2012).

PHD fingers, such as those found in PHF7, are best known for their ability to specifically bind histones that have been modified on their N-terminal tails, in particular methylated H3K4. This study shows that both Drosophila and human PHF7 can directly associate with dimethylated H3K4, indicating that PHF7 is indeed a histone code reader. It is uncommon for PHD domains to associate preferentially with H3K4me2 over H3K4me3, but this specificity has been observed previously, and is likely important for how PHF7 modulates expression of its targets. Both di- and trimethylated H3K4 are found at actively transcribed genes, but H3K4me2 is normally localized at the 5′ end of coding sequences, downstream of H3K4me3, which is near promoters. The two marks are also regulated by different demethylases. A few recent studies have started to dissect effects of H3K4me2 on gene transcription, but the exact mechanisms are not well understood. Some PHD finger proteins also contain other domains, such as those that modify histones enzymatically. This does not appear to be the case for PHF7, and the region of homology between PHF7 homologs of different species is restricted to the PHD domains. However, individual PHD fingers can bind modified histone tails independently, and it is yet unclear which PHD finger in PHF7 contacts H3K4me2 and what activities the others might have. The logic of how PHF7 is recruited to specific loci and affects chromatin structure and gene activity are interesting questions for future work (Yang, 2012).

Another point of interest is how a reader of such a common epigenetic mark would have a sex-specific role in regulating male germline identity. It has been observed that mutation of an H3K4me2 demethylase in Caenorhabditis elegans, which leads to increased dimethylation at H3K4, results in ectopic activation of male-specific germline genes (Katz, 2009). A similar mutation in Drosophila causes female germline developmental defects (Szabad, 1988), which may be related to the germline atrophy observed when PHF7 expression was upregulated in female germ cells. These data are consistent with the hypothesis that H3K4me2 has a role in regulating the male germline genome. Interestingly, another germline chromatin factor, No child left behind (NCLB), has been identifed that is expressed in germ cells of both sexes but required for GSC function only in males (Casper, 2011). Thus, NCLB may cooperate with PHF7 in regulating the male GSC transcriptional program (Yang, 2012).

Based on sequence homology, orthologs of Phf7 are present in a wide range of mammalian species. Human and mouse PHF7 share extensive homology to Drosophila PHF7 throughout the N-terminus where the PHD fingers are present, and the results confirm that human PHF7 recognizes H3K4me2, similar to the fly protein. Interestingly, EST profiling indicates strong testis biases for Phf7 expression in many species, including humans, mice, rats, and dogs. Moreover, several studies that performed genome-wide RNA profiling from purified mouse germline populations indicate that mouse Phf7 expression is present in spermatogonia and is further induced in spermatocytes. Remarkably, human PHF7 was able to rescue fecundity defects in male flies mutant for Phf7. Thus, the sequence conservation observed between mammalian and Drosophila Phf7 represents true functional orthology (Yang, 2012).

As in Drosophila, germline sex determination in mouse is regulated at an early stage and is controlled by important signals from the soma, which for the mouse include retinoic acid and FGF9. Such signals regulate the timing of meiotic entry, which is different between the sexes, and also influence sex-specific programs of germline gene expression, such as expression of the key male-specific factor nanos2. Significant changes in germ cell chromatin occur during this critical time in germ cell development, including changes in the H3K4 methylation state. Thus, Phf7 represents a prime candidate for interpreting these chromatin changes in a sex-specific manner to regulate male-specific gene expression. It will be of great interest to determine whether Phf7 plays a critical role in mouse and human male germ cell development, as is proposed for Drosophila (Yang, 2012).

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).

Germline masculinization by Phf7 in D. melanogaster requires its evolutionarily novel C-terminus and the HP1-family protein HP1D3csd

Germ cells in Drosophila melanogaster need intrinsic factors along with somatic signals to activate proper sexual programs. A key factor for male germline sex determination is PHD finger protein 7 (Phf7), a histone reader expressed in the male germline that can trigger sex reversal in female germ cells and is also important for efficient spermatogenesis. This study found that the evolutionarily novel C-terminus in Phf7 is necessary to turn on the complete male program in the early germline of D. melanogaster, suggesting that this domain may have been uniquely acquired to regulate sexual differentiation. Genes were sought that regulated by Phf7 related to sex determination in the embryonic germline by transcriptome profiling of FACS-purified embryonic gonads. One of the genes positively-regulated by Phf7 in the embryonic germline was an HP1 family member, Heterochromatin Protein 1D3 chromoshadow domain (HP1D3csd). This gene is needed for Phf7 to induce male-like development in the female germline, indicating that HP1D3csd is an important factor acting downstream of Phf7 to regulate germline masculinization (Yang, 2021).

This study investigated how Phf7 regulates sex determination in the embryonic germline, and one of the interesting findings is that the unusual C-terminus of Phf7 is necessary for its effects in germline masculinization. The N-terminus of Phf7 is a conserved module comprised of three zinc fingers, of which at least one is functionally essential, and this part of the Phf7 protein evolved from G2E3 (G2/M E3 Ubiquitin Ligase), a protein also made up of three zinc fingers. In contrast, the C-terminus of Phf7 is evolutionarily novel and is not similar to any known domains, suggesting that this domain is undergoing very rapid evolution, a feature not uncommon for factors involved in sex determination (Yang, 2021).

Previously a phylogenetic analysis of Phf7 proteins was conducted across the species tree, and surprisingly it was found that Phf7 in insects and amniotes do not share a common ancestor. Those findings with the latest results indicate that Phf7 in these two animal branches are not orthologous to each other, and that the emergence of the novel C-terminus is likely a unique event that occurred in Drosophila to regulate sexual differentiation in the germline. Recently, mouse Phf7 was demonstrated to be expressed in spermatocytes and can ubiquitinate histones to facilitate histone to protamine exchange. This shows that the expression patterns and functions of D. melanogaster and mouse Phf7 are different, albeit both acting on the male germline. These observations further suggest that the C-terminus of D. melanogaster PHF7 evolved onto an existing module of three zinc fingers, thereby creating new ways to regulating germline sexual development. This is a very interesting example that adds to the collection of diverse mechanisms in sex determination (Yang, 2021).

What does this uncommon C-terminus of PHF7 do? The two most intuitive ideas are that it acts as a transactivation domain like those found in transcription factors, or that it can recruit other effector molecules through protein-protein interactions. The former idea did not hold up when tested in S2 cells. The possibility that the Phf7 C-terminus acts as a bridge between its histone-associating N-terminus and other transcription factors or chromatin factors to alter target gene expression is an appealing one but there is currently no direct data that support this idea (Yang, 2021).

Downstream effectors of Phf7 were sought in the embryonic germline, and it was revealed that HP1D3csd is activated by Phf7 to regulate germline masculinization. Two different genetic tests were perfomed, and while both indicated that Phf7 and HP1D3csd genetically interact, there were some differences in the results. In the Phf7-induced female germline loss assay, both reduction and gain of HP1D3csd expression were found to be exacerbated the Phf7-induced phenotype. In comparison, in the spermatogenesis rescue assay, loss of one HP1D3csd copy hampered rescue whereas HP1D3csd overexpression enhanced spermatogenesis in XX germ cells. The latter experiment is a more direct assay of germline masculinization whereas germline loss can potentially be caused by secondary effects unrelated to sexual development. Therefore, it is thought the results of the spermatogenesis rescue experiments more accurately reflect the relationship between Phf7 and HP1D3csd. In addition to the transcriptome results, HP1D3csd has been identified along with Phf7 to be a part of sex-biased mechanisms in other contexts not limited to the germline. These also support the model that Phf7 and HP1D3csd function synergistically (Yang, 2021).

Phf7 regulates male germline development, and it can associate with the active histone mark methylated H3K47, but it is unclear what Phf7 then does to regulate expression of target genetic loci. H3K9 methylation has also been reported to be important for maintaining sexual differentiation programs in the Drosophila germline. The identification of HP1D3csd as an important downstream factor provides interesting new ideas regarding how the male germline program is initiated and regulated. CSDs have been shown to interact with various chromatin remodelers, thus one appealing model would be that Phf7 can activate or even recruit HP1D3csd to loci important for germline masculinization. This would in turn bring chromatin remodelers to such genes for expression activation and regulation and initiate male-development of the germline. Given the other finding in this study that the C-terminus of Phf7 is an essential part of this process, it would be very interesting to now study which of these factors interact and cooperate with one another (Yang, 2021).

Germline sex determination regulates sex-specific signaling between germline stem cells and their niche

Establishing germ cell sexual identity is critical for development of male and female germline stem cells (GSCs) and production of sperm or eggs. Germ cells depend on signals from the somatic gonad to determine sex, but in organisms such as flies, mice, and humans, the sex chromosome genotype of the germ cells is also important for germline sexual development. How somatic signals and germ-cell-intrinsic cues combine to regulate germline sex determination is thus a key question. This study found that JAK/STAT signaling in the GSC niche promotes male identity in germ cells, in part by activating the chromatin reader Phf7. Further, it was found that JAK/STAT signaling is blocked in XX (female) germ cells through the action of the sex determination gene Sex lethal to preserve female identity. Thus, an important function of germline sexual identity is to control how GSCs respond to signals in their niche environment (Bhaskar, 2022).

This study presents data that provides new insights into germline sex determination and the regulation of male versus female GSC identity. First, it was found that one key function of the JAK/STAT pathway in GSCs is to promote male identity and directly activate expression of the male germline chromatin regulator Phf7. Further, it was found that an important role for Sxl in female germ cells is to block the JAK/STAT pathway and prevent this signal from masculinizing the germline. Therefore, one key aspect of germline sexual identity is to regulate how GSCs respond to signals in their niche environment (Bhaskar, 2022).

Different findings have led to different conclusions about the role of the JAK/STAT pathway in male GSCs. When STAT activity is removed from individual GSCs, they are lost rapidly from the niche, indicating a role in GSC identity or maintenance. However, when STAT is removed from all GSCs, they exhibit defects in niche adhesion but can otherwise function as GSCs, although GSC loss is also observed. The JAK/STAT pathway has also been implicated in aging of GSCs and their niche. One interpretation of these diverse data would be that the JAK/STAT pathway is important for specific aspects of male GSC function, such as regulation of cell adhesion and the cell cycle, but it is not required for stem cell identity per se (Bhaskar, 2022).

A different role is proposed for the JAK/STAT pathway, which is to regulate GSC sexual identity. Previously it was reported that the JAK/STAT pathway is important for establishing male identity in the embryonic germline. This study shows that one defect observed in XX germ cells present in a male soma is that they exhibit reduced JAK/STAT signaling. Further, activation of the JAK/STAT pathway can partially rescue these XX germ cells, promoting a male identity and progression into spermatogenesis. Thus, it is proposed that the JAK/STAT pathway remains a key masculinizing signal for the germline throughout development and into adulthood. One possibility is that the JAK/STAT pathway regulates only GSC sex and that other roles, such as regulating a specific set of cell adhesion proteins, represent downstream consequences of altering sexual identity. Alternatively, the JAK/STAT pathway could regulate GSC sexual identity and other aspects of GSC behavior independently. One important way in which the JAK/STAT pathway promotes a male identity in the germline is by activating the male sex determination factor Phf7. Previously, it was shown that Phf7 is important for male identity in the germline and proper spermatogenesis. Phf7 likely promotes male germline identity by acting as a chromatin 'reader' and binding to histone H3 methylated at position K4. Phf7 is also toxic to female germ cells, making the sex-specific regulation of Phf7 extremely important. This study shows that the JAK/STAT pathway is a direct regulator of Phf7 expression in both embryos and adults. STAT protein can bind to the Phf7 locus, and consensus STAT binding sites near the male-biased promoter are essential for proper male expression of Phf7 and its ability to function in spermatogenesis. Expression from the male-biased promoter is important in part because the transcript from the downstream, 'female' promoter is subject to translational repression. Thus, Phf7 represents an important link between the JAK/STAT pathway and male identity in the germline (Bhaskar, 2022).

Sxl acts as a key regulator of sex determination in both the soma and the germline, and it is necessary and sufficient to confer female identity. However, the role of Sxl in the germline has remained mysterious. In the soma, Sxl regulates sexual identity through tra and dosage compensation through msl-2, but these genes do not play a role in the germline. Instead, this study has found that a key role of Sxl in the germline is to repress the JAK/STAT pathway in female germ cells (Bhaskar, 2022).

Initially, only the male somatic gonad expresses ligands for the JAK/STAT pathway and is capable of promoting JAK/STAT activation in the germ cells. However, ligands for the JAK/STAT pathway eventually become active in the germarium of the ovary, where they are important for the function or maintenance of the somatic escort cells. Sxl acts to repress JAK/STAT response in the female germ cells and thereby prevents activation of male-promoting factors such as Phf7. Somatic cells of the ovary such as the escort cells are still able to respond to these ligands and activate the JAK/STAT pathway, even though they also express Sxl. How Sxl is able to repress the JAK/STAT response in a germline-specific manner remains unknown, although the levels of Sxl appear higher in the GSCs than in the surrounding soma. However, the fact that an activated Hop (hop^Tum) can partially rescue the germline in XX males indicates that Sxl is repressing the pathway at the level of Hop or above. Interestingly, RNA for the JAK/STAT receptor domeless was identified in a pull-down experiment with Sxl, suggesting this could be a relevant target for regulation (Bhaskar, 2022).

These data support a model where the JAK/STAT pathway is important for activating male identity in the germline and expression of male genes such as Phf7, while this pathway is repressed in female germ cells by Sxl. Loss of Sxl from the female germline leads to both upregulation of JAK/STAT signaling and inappropriate expression of Phf7. In addition, suppression of the JAK/STAT pathway can partially rescue loss of Sxl in the female germline. Thus, regulation of the JAK/STAT pathway is one key aspect of how Sxl promotes female germline identity. However, no ability was observed for loss of Phf7 to rescue loss of Sxl from the female germline. This is in contrast to previously published results where loss of Phf7 was shown to rescue the female germline in sans fille mutants, which also primarily affects the germline by disrupting Sxl expression. This study has now reduced Phf7 function by both RNAi and using null Phf7 mutants, in both Sxl and sans fille loss-of-function backgrounds and observed no rescue or modification of the germline defects present. It is concluded that, while regulation of Phf7 by the JAK/STAT pathway and Sxl is clearly important for proper germline sexual development, ectopic expression of Phf7 is not the only defect present in Sxl mutant female germ cells; there must be additional targets for regulation by Sxl and JAK/STAT that are disrupted in Sxl mutants. In support of this view, loss of STAT from the male germline has a more severe phenotype than loss of Phf7. Previously, it has been shown that expression of another male-promoting factor in the germline, Tdrd5l, is regulated by Sxl. While this regulation appears to be, at least in part, via Sxl acting on the Tdrd5l mRNA to influence levels of Tdrd5l protein, it is possible that Tdrd5l is also regulated at the transcriptional level as an additional target of the JAK/STAT pathway (Bhaskar, 2022).

It is intriguing that Sxl acts as a negative regulator of the JAK/STAT pathway in both the soma and the germline but does so in different ways. In the soma, Sxl activates an alternative splicing cascade that leads to splicing of dsx in the female mode, creating the DSXF protein, while the DSXM protein is produced in males by default. An important sex-specific trait in the embryonic gonad is that male somatic cells produce ligands for the JAK/STAT pathway that activate JAK/STAT signaling specifically in male germ cells, and this is regulated in a manner dependent on dsx. Thus, in addition to being a negative regulator of JAK/STAT signal reception in the germline, Sxl acts as a negative regulator of JAK/STAT ligand production in the soma. Together, these independent aspects of regulation by Sxl combine to ensure that the masculinizing effects of the JAK/STAT pathway are restricted to male germ cells (Bhaskar, 2022).

An important conclusion from this work is that germline sex determination regulates how GSCs communicate with their surrounding stem cell niche. Germline sex determination is regulated by both germline autonomous cues, based on the germline sex chromosome constitution, and non-autonomous signals from the soma. The autonomous cues, acting through Sxl, regulate how signals from the niche are received and interpreted by the GSCs. In both the testis and ovary GSC cell niches, the JAK/STAT pathway is important for regulating somatic cells like the cyst stem cells in the testis and the escort cells in the ovary. However, this pathway is only required in the male GSCs and not female GSCs. It is proposed that it is essential to block JAK/STAT signaling in female GSCs to prevent their exposure to this masculinizing signal. Indeed, activation of the JAK/STAT signal is sufficient to promote male identity in XX germ cells, and removal of STAT is sufficient to partially rescue the defects observed in XX germ cells that have lost Sxl. Thus, a key aspect of how Sxl promotes female identity in the germline is to prevent female GSCs from being masculinized by activators of the JAK/STAT pathway present in the niche environment (Bhaskar, 2022).

It is important to note that, when the sex chromosome genotype affecting germline is referred to as 'sex determination' this could result from any contribution of sex chromosome genotype to successful spermatogenesis or oogenesis. For example, if dosage compensation is incomplete or non-existent in the germline, then the presence of two X chromosomes will lead to increased X chromosome gene expression, which may be incompatible with male germline differentiation. Similarly, a single X chromosome dose may be incompatible with oogenesis. It is also possible that the number of X chromosomes present in the germline has additional affects besides the presence or absence of Sxl expression. While XX germ cells present in a male soma exhibit severe atrophy and loss, the expression of Sxl in the male germline has a much weaker phenotype. Thus, there may be additional consequences of sex chromosome genotype on germline function beyond that which is controlled by Sxl. A better understanding of what germline sexual identity means in Drosophila, in particular at the level of whole-genome gene expression levels, is required before it will be possible to assess the true contribution of germline sex chromosome constitution to germline sex determination. Further, how the effects of X chromosome number on germline sexual development in Drosophila relate to infertility observed in patients with disorders of sexual development such as Klinefelter's and Turner's syndromes remains to be investigated (Bhaskar, 2022).

One limitation of this study is the relatively low frequency with which it was possible to rescue the XX germline in tra mutants by downregulating Sxl or upregulating the JAK/STAT pathway. This could be due to technical limitations of the timing or level of expression of the reagents used. Alternatively, this could mean that there is another interesting defect in XX germ cells present in a male somatic environment besides that is caused by expression of Sxl and downregulation of the JAK/STAT pathway. Another limitation of the study is that the molecular target for Sxl in regulating the JAK/STAT pathway remains unknown. RNA-seq analysis of Bam mutant testes and ovaries suggested that hop RNA splicing is differentially regulated between males and females. However, extensive experimental analysis failed to reveal a role for Sxl in regulating hop in the germline. Thus, this remains an important area for future research (Bhaskar, 2022).

Search PubMed for articles about Drosophila Phf7

Bhaskar, P. K., Southard, S., Baxter, K. and Van Doren, M. (2022). Germline sex determination regulates sex-specific signaling between germline stem cells and their niche. Cell Rep 39(1): 110620. PubMed ID: 35385723

Casper, A. L., Baxter, K. and Van Doren, M. (2011). no child left behind encodes a novel chromatin factor required for germline stem cell maintenance in males but not females. Development 138: 3357-3366. PubMed ID: 21752937

Katz, D. J., Edwards, T. M., Reinke, V. and Kelly, W. G. (2009). A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory. Cell 137: 308-320. PubMed ID: 19379696

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

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

Yang, S. Y., Baxter, E. M. and Van Doren, M. (2012). Phf7 controls male sex determination in the Drosophila germline. Dev Cell 22: 1041-1051. PubMed ID: 22595675

Yang, S. Y. (2021). Germline masculinization by Phf7 in D. melanogaster requires its evolutionarily novel C-terminus and the HP1-family protein HP1D3csd. Sci Rep 11(1): 6308. PubMed ID: 33737548

date revised: 23 June 2023

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