Author response:
The following is the authors’ response to the original reviews.
Public Review:
Reviewer #1 (Public review):
Leukemia-driving NUP98 oncofusion proteins form chromatin-associated biomolecular condensates in the nucleus, and these structures are important for oncogenic transformation. Most NUP98 fusions do not contain domains that mediate the recognition of specific DNA elements. Instead, they entail domains that are important for chromatin regulation. For instance, the NUP98::KDM5A fusion features a fusion of the NUP98 N-terminus with the third PHD domain of the histone demethylase KDM5A. As PHD domains are critical for the recognition of methylated histones without any sequence specificity, it is not clear what controls the condensation and chromatin binding of NUP98::KDM5A, leading to the induction of oncogenic transcriptional programs.
In this work, the authors use a combination of cellular and in vitro studies to show that biomolecular condensation of NUP98::KDM5A is dependent on H3K4me3 binding. Their model proposes that concentration-dependent chromatin-associated condensation of NUP98::KDM5A depends on local densities of H3K4me3 on chromatin and the levels of the fusion oncoprotein. In line with this, the analysis of gene expression data from NUP98::KDM5A-positive AML cells shows a positive correlation between differentially expressed genes and H3K4me3 levels.
This is an interesting manuscript that aims to dissect the molecular mechanisms underlying biomolecular condensation of the NUP98::KDM5A oncoprotein. The work is solid, and the results are well explained and presented in a logical order. However, the study suffers from several weaknesses that if addressed would improve the study.
Major points:
(1) All cellular experiments are performed in settings of transient transfection of NUP98::KDM5A in non-hematopoietic cell types. These conditions are not physiologically relevant, as these cells do not depend on the fusion oncogene. Therefore, any claims about concentration-dependent effects on condensation need to be validated in AML cells that are driven by NUP98::KDM5A. While this may not be possible in primary patient-derived cells, several groups have published AML models of NUP98::KDM5A-driven AML that could be used.
We appreciate the reviewer’s point regarding physiological relevance. Our goal in these cellular microscopy experiments was to dissect the biophysical principles of NUP98::KDM5A (including its mutants) condensate formation under controlled expression levels (concentration). While AML model systems driven by NUP98::KDM5A are available, they do not offer this possibility because of pre-existing NUP98::KDM5A expression. Suspension culture of hematopoietic cells also poses practical challenges for correlative FISH+IF and high-resolution microscopy analysis. We agree that validating our observed behaviors in AML models would be valuable, e.g. by creating HSPC lines with inducible expression of tagged NUP98::KDM5A and its mutants, but such experiments fall outside the scope of the current study. We will add text acknowledging this limitation and clarifying that our mechanistic conclusions are grounded in biophysical principles that should generalize across cell types.
In the revised manuscript, we have added a section on study limitations at the end of the discussion section:
Study limitations: Cellular experiments in this study were performed using transient transfection of WT NUP98::KDM5A and its mutants in HEK293 and U2OS cells. While these non-hematopoietic cell models limit physiological relevance, they allowed for controlled expression levels in the absence of endogenous oncofusion and the ease of imaging, enabling investigation of the biophysical principles of condensate formation, the main objective of our study.
(2) The results presented in Figure 4 are not entirely supportive of the mechanism. It is known that active gene expression correlates with high H3K4me3 levels; therefore, the correlations shown by the authors are expected. Yet, the authors do not discuss the fact that many H3K4me3-positive genomic regions do not show NUP98::KDM5A binding. This should be elaborated on in the discussion section.
We agree that active transcription correlates with H3K4me3, and that this baseline relationship must be considered. Our analysis explicitly uses fold-change between patient cells and healthy controls as the readout. This comparison inherently accounts for the activating effect of H3K4me3 itself. Regarding the reviewer’s comment that “many H3K4me3-positive genomic regions do not show NUP98::KDM5Abinding”, we would like to clarify that this point is exactly what our manuscript aims to explain. Our cell line studies show that, at patient-relevant expression levels, NUP98::KDM5A condensates form preferentially at H3K4me3 loci with high local mark density. Considering that NUP98::KDM5A concentration in the nucleus is lower than the KD between KDM5A PHD3 and H3K4me3, this means that H3K4me3 loci with lower mark density will not see NUP98::KDM5A binding without the high local concentration of the fusion protein (as a result of condensate formation). This is consistent with our observation that genes with the highest local density show disproportionately stronger upregulation.
We have modified the final paragraph of the section describing our analysis of gene expression in patient samples to add:
Importantly, to account for the activating effect of H3K4me3 itself, our analysis explicitly uses fold-change between patient cells and healthy controls as the readout.
(3) While the focus of the manuscript is on NUP98::KDM5A, this oncofusion is part of a family of >30 fusions that join the NUP98 N-terminus to a variety of factors with roles in epigenetic control and transcription. While the repertoire of NUP98 fusion partners is diverse with regard to functional domains, they all induce a conserved set of target genes that is characteristic of this leukemia subtype. How can this be achieved in the context of NUP98 fusion proteins that do not contain a PHD domain, such as NUP98::NSD1 or NUP98::HOXA9? Please discuss this.
We appreciate this important conceptual question. Many NUP98 fusions, despite diverse partner domains, produce similar transcriptional programs. Our current hypothesis is that the highly active status of the HOX cluster genes in HSPC attracts NUP98 oncofusions targeting H3K4me3 (e.g., KDM5A and PHF23), while NUP98::HOXA9 and other transcription factor fusions directly target the HOX cluster via DNA sequence recognition. This convergence and the broad targets of the dysregulated HOX transcription factors explain the transcriptional program similarity, sustained by the previously described enrichment of transcriptional co-activators by NUP98 oncofusion condensates. Of note, while several fusion partners of NUP98 lack PHD domains, the NUP98::NSD1 oncofusion includes several PHD domains of NSD1, which have previously been suggested to be important for leukemogenesis (PMID: 17589499). The discussion section has been modified to add:
We envision that a similar mechanism targets other NUP98 fusions containing H3K4me3-recognizing reader domains (e.g., NUP98::PHF23) to leukemogenic loci, complementing direct targeting of the HOX genes through DNA sequence recognition in fusions containing DNA-binding domains (e.g., NUP98::HOXA9).
Reviewer #2 (Public review):
In this manuscript, the authors investigate how the oncogenic fusion protein NUP98KDM5Aalters gene expression in leukemia, using a combination of cellular experiments with model and patient cell lines, as well as in vitro studies. Upon transfection of U2OS cells with mEGFP-tagged NUP98-KDM5A, the authors show that the fusion proteins form sub-micrometre puncta, whereas KDM5A alone does not. These foci are also observed at expected native expression levels (using OpenCell data). The tag has an effect here, as switching to an mCherry tag raises the apparent saturation concentration for phase separation. Finally, the authors show via super-resolution imaging that the foci correlate with H3K4me3 distribution.
In vitro, the fusion protein forms amorphous, gel-like condensates at double-digit nanomolar concentrations. Truncation analysis identifies PHD3 of KDM5A as required for maximal phase separation, consistent with the ability of the protein to bind H3K4me3 peptides. Addition of polynucleosomes increases the amount of fusion protein partitioning into the condensate in anH3K4me3-binding-dependent manner. Condensates are gel-like with slow internal dynamics in vitro; in cells, however, the dynamics depend on the position of the EGFP tag (no corresponding experiments with mCherry are shown). Reconstitution with H3K4me3- and H3K4me0-modifiedarrays shows colocalization with both wild-type NUP98-KDM5A and the binding mutant. Here, H3K4me3 arrays recruit ~20% more protein and yield gel-like structures in a manner dependent on the PTM and on the PHD finger.
In cells, the fusion protein colocalizes with H3K4me3-marked loci, including the HOX clusters, as confirmed by FISH. Finally, re-analysis of published expression datasets from patient cells shows that genes are predominantly upregulated and that the upregulated genes are H3K4me3-marked.
This is a well-executed mechanistic study. The data convincingly establish that NUP98KDM5A forms sub-micrometre foci at realistic expression levels, that these foci correlate with H3K4me3-marked sites, that the PHD3-H3K4me3 interaction mediates chromatin binding while the NUP98moiety drives phase separation in vitro, that foci in cells overlap genes heavily decorated with H3K4me3, and that H3K4me3-marked genes are those found to be upregulated in patient datasets. These are important mechanistic findings and of interest to the community.
Still, the functional/causal link is a bit more tentative, as the data is mostly correlative, since it is not directly established that there is feedback between H3K4 methylation, NUP98KDM5Arecruitment, phase separation and target gene overexpression. An experiment that could further bolster this claim would be a direct test of whether NUP98-KDM5A expression drives overexpression of bound genes, e.g. expression of the fusion protein vs PHD- and NUP98-mutant variants, followed by qPCR of target genes, such as the HOX cluster, and possibly H3K4me3 ChIP at the same loci. As all the constructs and cell lines exist, this could be feasible and would substantially strengthen the manuscript.
We thank the reviewer for the thoughtful evaluation and agree that the causal link betweenH3K4me3 recognition, condensate formation, and gene activation remains partly correlative. Experiments such as qPCR or ChIP following expression of WT versus mutant constructs would indeed strengthen the causal chain. However, performing these assays across multiple constructs and loci in a physiologically relevant system is not feasible within the current revision cycle. We have added text acknowledging this limitation and clarifying that our study focuses on establishing the biophysical mechanism of targeting, while functional consequences are inferred from patient datasets rather than new perturbation experiments.
We modified the Study limitations section of the Discussion to add:
Similarly, our study focuses on the biophysical mechanism of chromatin targeting by the NUP98::KDM5A oncofusion, with functional consequences inferred from patient-derived datasets rather than defined perturbation experiments.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Figure 1 - Figure Supplement 2: show co-localisation of large droplets of the chromatin-binding mutant with nucleoli by co-immunostainings, using nucleolar markers.
(2) Figure 1E: Show another histone mark as a control, ideally H3K27me3.
(3) The authors are encouraged to stick to the recommended nomenclature of fusion genes(https://www.nature.com/articles/s41375-021-01458-0).
Reviewer #2 (Recommendations for the authors):
(1) The phase-separation behaviour clearly depends on the fusion FP, as the switch from mEGFP to mCherry shifts the apparent saturation concentration to ~200 nM. The authors should discuss, and where possible test, how much of the observed behaviour is attributable to the FP itself: what happens in the absence of any FP, and which construct (mCherry- or mEGFP-tagged) the authors consider the better proxy for the untagged fusion protein.
(2) This is particularly relevant given that the in-cell dynamics (FRAP) are reported to depend on the position of the EGFP tag, and no corresponding mCherry experiments are shown.
(3) The authors claim a patient NUP98-KDM5A concentration of ~180 nM. This is based on data for NUP98 in HEK293 cells, with the added correction factor for having only one allele mutated in patients. Still, the direct jump from HEK293 to patient leukemia cells is big; so the authors should formulate this more carefully: no conc. determination was done directly in patients.
(4) Chicken erythrocyte chromatin: What is the endogenous H3K4me3 level in the chicken erythrocyte chromatin used here?
(5) Figure 1 - Figure Supplement 3B. The legend defines the square as the mean but does not explain the remaining symbols (vertical lines, horizontal lines, grey versus black dots). Please define these plot elements.
(6) In the reconstitution experiments, the arrays and the condensates appear to colocalize only weakly, and, a bit counterintuitively, the strongest apparent colocalization is seen for the NUP98KDM5A binding mutant. Can the colocalization be quantified and interpreted?
(7) Figure 2 - Figure Supplement 3. Raising the chromatin concentration from 12.5 to 100 nM produces little visible change in the images. Where does the additional chromatin go? Does it stay in solution? As in point 4, the best colocalization again appears to be obtained with the mutant, which is unexpected and a bit difficult to reconcile with the proposed H3K4me3-PHDdrivenrecruitment model.
(8) That highly expressed genes in patients carry H3K4me3 is basically expected and is not by itself informative. The relevant question is the origin of the effect: is H3K4me3, and the associated upregulation, a consequence of NUP98-KDM5A condensate formation? Analyzing the direct effects of NUP98-KDM5A (and mutants) on specific target gene expression profiles could help to dissect this further.
We thank the reviewers for these recommendations. We have made the following revisions to the manuscript:
We replaced NUP98-KDM5A with NUP98::KDM5A, in accordance with the recommended nomenclature. We made this change in both the text and the figures. We also corrected other NUP98 on confusions to reflect this recommendation.
We have revised Figure 1 – Figure Supplement 3B and the accompanying legend to describe the symbols used in this figure.
