SUMOylation of Bonus, the Drosophila homolog of Transcription Intermediary Factor 1, safeguards germline identity by recruiting repressive chromatin complexes to silence tissue-specific genes

Reviewer #1 (Public Review):

Summary:
This important study from Godneeva et al. establishes a Drosophila model system for understanding how the activity of Tif1 proteins is modified by SUMO. The authors nicely show that Bonus, like homologous mammalian Tif1 proteins, is a repressor, and that it interacts with other co-repressors Mi-2/NuRD and setdb1 in Drosophila ovaries and S2 cells. They also show that Bonus is SUMOylated by Su(var)2-10 on at least one lysine at its N-terminus to promote its interaction with setdb1. By combining nice biochemistry with an elegant reporter gene approach, they show that SUMOylation is important for Bonus interaction with setdb1, and that this SUMO-dependent interaction triggers high levels of H3K9me3 deposition and gene silencing. While there are still major questions of how SUMO molecularly promotes this process, this study is a valuable first step that opens the door for interesting future experimentation.

Major Point:
The RNAseq and ChIPseq data is not available. This is critical for the review of the paper and would help the readers and reviewers interpret the Bonus mutant phenotype and its mechanism of repressing genes.

1. The author's conclusion that Bonus SUMOylation is "essential for its chromatin localization" is not supported by the data. Figure 5F shows less 3KR mutant in the chromatin fraction but there is still significant signal.
2. The author's conclusion that Bonus is SUMOylated at a single site close to its N-terminus is not necessarily true. In several SUMO and Bonus blots throughout the paper (5B, 6C, S4A), there are >2 differentially migrating species that could represent more than one SUMO added to Bonus. While the single K20R mutation eliminates all of these species in Fig 5C, it is possible that K20R SUMOylation is required for additional SUMOylation events on other residues. One way to determine if Bonus is SUMOylated on multiple sites is to add recombinant SUMO protease to the extract and see if multiple higher molecular weight bands collapse into a single migrating species (implying multiple SUMOs) or multiple migrating species (implying something else is altering gel migration).
3. The authors state that most upregulated genes in BonusGLKD are not highly enriched in H3K9me3. The heatmap in figure 3D is not an ideal presentation of this argument. The authors should show an example of what the signal on a highly enriched gene looks like for comparison. The authors also argue that because most upregulated genes in BonusGLKD are not highly enriched in H3K9me3, they must be indirectly repressed. Another possibility is that bonus-mediated H3K9me3 is only important (and present) during early nurse cell differentiation and is later lost and dispensable during the rapid endocycles. After bonus establishes repression though H3K9me3, it might be maintained through bonus-Mi2/Nurd, something else, or nothing at all. The authors could discuss this possibility or perform H3K9me3 ChIP during cyst formation and early nurse cell differentiation rather than in whole ovaries, which are enriched for later stages.
4. The BonusGLKD RNAseq analysis is underwhelming. The conclusion that "Bonus represses tissue-specific genes" has limited value. Every gene that is not expressed in ovaries is "tissue-specific." What subset of tissue-specific genes does Bonus repress? What common features do these genes have and how do they compare to other sets of tissue-specific genes, such as those reportedly repressed by setdb1, Polycomb proteins, small ovary, l(3)mbt, and stonewall (among others in female germ cells). Comparing these available data sets could help the authors understand the mechanism of Bonus repression and how BonusGLKD leads to sterility. The authors could also further analyze the differences between nos-Gal4 and MT-Gal4 to better understand why nos- but not MT-driven knockdown is sterile.

Main Study Limitations:

1. It is unclear which genes are directly vs indirectly regulated by bonus, which makes it difficult to understand Bonus's repressive mechanism. Several lines of experiments could help resolve this issue. 1) Bonus ChIPseq, which the authors mentioned was difficult. 2) RNAseq of BonusGLKD rescued with KR3 mutation. This would help separate SUMO/setdb1-dependent regulation from Mi-2 dependent regulation. Similarly, comparing differentially expressed genes in Su(var)2-10GLKD, setdb1GLKD, 3KR rescue, and MI-2 GLKD could identify overlapping targets and help refine how bonus represses subsets of genes through these different corepressors.

2. The paper falls short in discussing how SUMO might promote repression. This is important when considering the conservation (of lack thereof) of SUMOylation sites in Tif1 proteins in distantly related animals. One piece of data that was not discussed is the apparent localization of SUMOylated bonus in the cytoplasmic fraction of the blot in Figure 5F. Su(var)2-10 is mostly a nuclear protein, so is bonus SUMOylated in the nucleus and then exported to the cytoplasm? Also, setdb1 is a nuclear protein, so it is unlikely that the SUMOylated bonus directly interacts with setdb1 on target genes. Together with Fig 5E (unSUMOylatable Bonus aggregates in the nucleus), one could make a model where SUMO solubilizes bonus (perhaps by disassembling aggregates) and indirectly allows it to associate with setdb1 and chromatin. It is also important to note that in Figure 5I, the K3R mutation appears to lessen but not eliminate Bonus interaction with setdb1. This data again disfavors a model where SUMO establishes an interaction interface between setdb1 and Bonus. To determine which form of Bonus interacts with setdb1, the authors could perform a setdb1 pulldown and monitor the SUMOylation state of coIPed Bonus through mobility shift. If mostly unSUMOylated bonus interacts with setdb1, and SUMO indirectly promotes Bonus interaction with setdb1 (perhaps by disassembling Bonus aggregates), then the precise locations of Bonus SUMOylation sites could more easily shift during evolution, disfavoring the author's convergent evolution hypothesis.

Reviewer #2 (Public Review):

Summary:
The authors analyze the functions and regulation of Bon, the sole Drosophila ortholog of the TIF1 family of mammalian transcriptional regulators. Bon has been implicated in several developmental programs, however the molecular details of its regulation have not been well understood. Here, the authors reveal the requirement of Bon in oogenesis, thus establishing a previously unknown biological function for this protein. Furthermore, careful molecular analysis convincingly established the role of Bon in transcriptional repression. This repressor function requires interactions with the NuRD complex and histone methyltransferase SetDB1, as well as sumoylation of Bon by the E3 SUMO ligase Su(var)2-10. Overall, this work represents a significant advance in our understanding of the functions and regulation of Bon and, more generally, the TIF1 family. Since Bon is the only TIF1 family member in Drosophila, the regulatory mechanisms delineated in this study may represent the prototypical and important modes of regulation of this protein family. The presented data are rigorous and convincing. As discussed below, this study can be strengthened by a demonstration of a direct association of Bon with its target genes, and by analysis of the biological consequences of the K20R mutation.

Strengths:
1. This study identified the requirement for Bon in oogenesis, a previously unknown function for this protein.
2. Identified Bon target genes that are normally repressed in the ovary, and showed that the repression mechanism involves the repressive histone modification mark H3K9me3 deposition on at least some targets.
3. Showed that Bon physically interacts with the components of the NuRD complex and SetDB1. These protein complexes are likely mediating Bon-dependent repression.
4. Identified Bon sumoylation site (K20) that is conserved in insects. This site is required for repression in a tethering transcriptional reporter assay, and SUMO itself is required for repression and interaction with SetDB1. Interestingly, the K20-mutant Bon is mislocalized in the nucleus in distinct puncta.
5. Showed that Su(var)2-10 is a SUMO E3 ligase for Bon and that Su(var)2-10 is required for Bon-mediated repression.

Weaknesses:
The study would be strengthened by demonstrating a direct recruitment of Bon to the target genes identified by RNA-seq. Given that the global ChIP-seq was not successful, a few possibilities could be explored. First, Bon ChIP-qPCR could be performed on the individual targets that were functionally confirmed (e.g. rbp6, pst). Second, a global Bon ChIP-seq has been reported in PMID: 21430782 - these data could be used to see if Bon is associated with specific targets identified in this study. In addition, it would be interesting to see if there is any overlap with the repressed target genes identified in Bon overexpression conditions in PMID: 36868234.

The second area where the manuscript can be improved is to analyze the biological function of the K20R mutant Bonus protein. The molecular data suggest that this residue is important for function, and it would be important to confirm this in vivo.