InteractiveFly: GeneBrief

identity crisis: Biological Overview | References

H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Genetic studies were used to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But the finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, it was discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDC's role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, these findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming (Shapiro-Kulnane, 2022).

Gene silencing is critical to establishing and maintaining cell fates. Once made, the decision to silence a gene is fortified by the acquisition of repressive histone modifications. While cell type specific epigenetic silencing is primarily associated with tri-methylation of histone H3 lysine (H3K27me3)-marked chromatin, tissue specific genes can also be repressed by H3K9 methylation. For example, in S. pombe, discrete H3K9me3 domains silence meiotic genes in vegetative cells. In C. elegans, H3K9 methylation silences inappropriate cell type specific genes, including germline genes, in somatic cells. In the D. melanogaster female germline, H3K9me3 silences male germline genes. In the mouse, H3K9me3 silences germline genes during early embryonic development. Studies carried out in mammalian tissue culture systems further identify H3K9me3-mediated gene silencing as a conserved and vital strategy for maintaining cell fates in a wide range of tissues). However, the molecular mechanisms controlling tissue specific installation of this epigenetic modification onto protein-coding genes are largely unknown (Shapiro-Kulnane, 2022).

The repressive H3K9me3 histone modification has well characterized roles in constitutive heterochromatin formation, and the transcriptional silencing of repetitive DNA elements such as satellite repeats and transposable elements (TEs). These studies identified two mechanisms of H3K9me3 recruitment. One mechanism involves small RNAs that guide localization through a complementary base pairing mechanism. In Drosophila and mammals, for example, the PIWI-associated small RNAs (piRNAs) guide the H3K9me3 silencing machinery to TEs. A second mechanism involves sequence specific DNA binding proteins. In mammals, for example, H3K9me3 can be guided to TEs by members of the vertebrate specific Kruppel-associated box zinc finger (KRAB-ZNF) family of DNA binding proteins. An analogous mechanism might exist in Drosophila, as two zinc finger proteins, KIPFERL and SMALL OVARY, were recently shown to have roles in TE silencing. Whether installation of this epigenetic modification onto protein-coding genes employs similar mechanisms remains unclear (Shapiro-Kulnane, 2022).

In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate PHD finger protein 7 (phf7) transcription. Thus, phf7 regulation in the female germline provides a powerful system to investigate how the H3K9me3 silencing mark is installed at protein-coding genes. phf7 encodes a predicted chromatin reader, first identified in a screen for genes expressed in male but not female embryonic germ cells. Curiously, phf7 is not essential for male fertility, but it is critical that female germ cells not express the PHF7 protein. Forced expression of PHF7 activates a toxic gene expression program enriched for genes usually restricted to the male germline. Even though PHF7 protein is limited to male germ cells, phf7 is transcribed in both male and female germ cells. Sex specificity is achieved by alternative transcription start sites (TSS). In testes, transcription from the upstream TSS (TSS1) produces a long mRNA isoform that makes protein (Shapiro-Kulnane, 2022).

In ovaries, an H3K9me3 mini domain prevents the selection of TSS1. Instead, transcription initiates from the downstream TSS (TSS2) to produce a short mRNA isoform that is not translated and has no function. Of the three Drosophila enzymes known to methylate H3K9, only SETDB1 has a specific and nonredundant role in repressing phf7. Germ cell specific loss of SETDB1, its binding partner ATF7IP, or the H3K9 reader HP1a produced ovarian germ cell tumors that inappropriately express the PHF7 protein. Notably, derepression results from losing the H3K9me3 mini domain. While these results establish the H3K9me3 mini domain controls phf7 transcription, the mechanisms controlling installation of this epigenetic modification remains unclear (Shapiro-Kulnane, 2022).

This study addresses the mechanism of targeted H3K9me3 deposition onto phf7 by identifying essential cis-regulatory elements and trans-acting factors. It was established that the required cis-regulatory sequences are conserved across Drosophila species, and the mechanism governing phf7 regulation was found to be different from what has been described for piRNA-guided H3K9me3 deposition on TEs. Lastly, it was discovered that repression depends on the previously unknown gene, CG4936, that has been named identity crisis (idc). idc encodes a zinc finger associated domain (ZAD) containing C2H2 zinc finger protein. Notably, loss of idc in germ cells interferes with phf7 repression by reducing H3K9me3 deposition. Together with the observation that IDC localizes to a conserved region within the phf7 gene, this analysis supports a model in which IDC guides H3K9me3 installation, thereby preventing accidental female-to-male programming (Shapiro-Kulnane, 2022).

SETDB1-controlled H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage-inappropriate phf7 transcription. SETDB1 is also required for TE silencing, where a piRNA-guided mechanism guides H3K9me3 deposition. This work establishes that phf7 is silenced by a piRNA-independent mechanism, and it was discovered that regulation depends on IDC, an uncharacterized member of the ZAD-ZNF family of DNA binding proteins. Regulation appears direct, as IDC is required for H3K9me3 deposition and localizes to the conserved first exon of phf7 in ovarian extracts. Collectively, these data establish that the sequence specific DNA binding protein IDC directs the H3K9 methylation machinery to build a silencing domain at the phf7 locus, thereby preventing accidental female-to-male reprogramming. In addition to extending understanding of how female germ cell fate is maintained, these studies provide the first example of a ZAD-ZNF protein guiding H3K9me3-mediated gene silencing (Shapiro-Kulnane, 2022).

Although this work is consistent with a simple model in which the SETDB1 H3K9me3 methyltransferase is recruited to phf7 by IDC, the mechanism by which IDC guides the methylation machinery to phf7 remains an open question. For example, it remains unclear whether recruitment is direct, as attempts to co-immunoprecipitate SETDB1 and IDC were unsuccessful. Furthermore, while this study established that IDC is required for H3K9me3 recruitment, chromatin transgenic reporter assays show that the region to which it binds, the conserved first exon, is not sufficient. This observation, together with identification of a second conserved cis-regulatory element within the adjoining intron invites speculation that IDC works in conjunction with other sequence-specific recruitment factors. One attractive contender is stonewall (stwl). STWL is a heterochromatin-associated protein that acts as a transcriptional repressor in vitro, associates with SETDB1 in yeast and localizes to the phf7 locus in S2 cells. Importantly, loss of stwl in ovaries leads to the inappropriate expression of the testis phf7 transcript. Future studies focused on the rules that govern female-specificity may reveal a general mechanism for context-dependent establishment of H3K9me3 silencing domains (Shapiro-Kulnane, 2022).

Previous work established that the H3K9me3 reader protein, HP1a, is essential for phf7 silencing. A requirement for HP1a is not surprising, as HP1a drives chromatin compaction and transcriptional silencing. HP1a also recruits H3K9 methyltransferases, enabling the spreading of the H3K9me3 repressive domain through a positive feedback mechanism. The spread of H3K9me3 over the three kb region within the phf7 gene likely occurs by an analogous mechanism. At the phf7 locus, however, the H3K9me3 domain does not extend into the open reading frame or the neighboring genes. Therefore, more work is needed to understand what stops the spreading of this repressive chromatin domain (Shapiro-Kulnane, 2022).

The SETDB1-controlled H3K9 methylation silencing pathway in female germ cells is not restricted to phf7. Genome-wide H3K9me3 profiling in wild-type and mutant ovaries has shown that SETDB1 silences two classes of protein-coding genes. One type includes genes usually expressed in the testis. A second class includes genes that are typically expressed in undifferentiated female germ cells but are silenced once the oocyte is specified, such as ribosomal protein S19b. These few examples of context dependent H3K9me3 gene silencing suggest a finely tuned guidance mechanism in the female germline. It will be interesting to explore whether other members of the ZAD-ZNF gene family serve as H3K9me3 guidance factors (Shapiro-Kulnane, 2022).

In summary, by focusing on a single biologically relevant gene, this study discovered a putative DNA binding protein that guides the installation of a H3K9me3 repressive domain onto a protein-coding gene. These findings are reminiscent of how TEs can be silenced in mammals wherein members of the KRAB-ZNF protein family recruit SETDB1 to establish epigenetic repression. Interestingly, the KRAB-ZNFs are vertebrate specific, and the ZAD-ZNFs are insect specific. Yet, both gene families exhibit similar patterns of species-specific gene expansion and diversification. These observations have fueled the speculation that ZAD-ZNFs and KRAB-ZNFs perform similar functions. In fact, IDC's closest human relative is ZNF75D, a KRAB-ZNF protein of unknown function. Whether this or other members of the KRAB-ZNF protein family are required for tissue specific H3K9me3 gene silencing will be an exciting area of further exploration (Shapiro-Kulnane, 2022).

Search PubMed for articles about Drosophila Identity crisis

Shapiro-Kulnane, L., Selengut, M. and Salz, H. K. (2022). Safeguarding Drosophila female germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein. PLoS Genet 18(12): e1010568. PubMed ID: 36548300

date revised: 5 August 2024

Home page: The Interactive Fly © 2026 Thomas Brody, Ph.D.