Nucleolar migration regulates meiotic sex chromosome inactivation via phase separation during mammalian spermatogenesis

Domain: neuroscience
Published: 2026-03-26
Editorial outcome: major_revision

Abstract
During spermatogenesis, the unsynapsed XY chromosomes undergo meiotic sex chromosome inactivation (MSCI) and form a heterochromatic XY body. Defects in MSCI lead to meiotic arrest and male infertility. Although DNA damage response (DDR) factors are established as key initiators of MSCI, how transcriptional silencing is subsequently achieved remains elusive. Here, we identify the nucleolar components NPM1, SENP3, and rRNA as essential downstream effectors of DDR signaling in MSCI. During pachytene, these components migrate to and transiently cover the XY body during MSCI establishment, before becoming restricted to a corner of the XY body. Genetic deletion of Npm1 or Senp3, or inhibition of rRNA transcription severely impairs MSCI. Mechanistically, SENP3-mediated deSUMOylation of NPM1 promotes its interaction with rRNA, enabling liquid-liquid phase separation, via which they exclude Pol II from the XY body. Together, these data reveal a critical role of nucleolar components in the transcriptional regulation of MSCI in mammalian spermatogenesis.

Introduction
In sexually reproducing organisms, meiosis is a specialized form of cell division that generates haploid gametes from diploid germ cells1. During spermatogenesis, homologous chromosome pairing and recombination occur at meiotic prophase I, which can be divided into five substages based on the chromosome configuration, i.e., leptotene (LEP), zygotene (ZYG), pachytene (PAC), diplotene (DIP), and diakinesis. Subsequently, spermatocytes undergo two successive cell divisions to produce round haploid spermatids, which then undergo a series of sequential programmed transitions including the histone-to-protamine exchange, condensation of the nucleus, and elongation, ultimately maturing into spermatozoa.

Unlike autosomes, the heteromorphic XY chromosomes are largely unsynapsed at meiotic prophase I, except for the pseudoautosomal region (PAR), and they form a membrane-less microscopically distinct spherical structure close to the nuclear periphery, termed the XY body. The unsynapsed XY chromosomes are transcriptionally silenced in the pachytene spermatocytes (PACs), which is referred to as meiotic sex chromosome inactivation (MSCI) and is essential for normal meiosis and male fertility2, 3.

Distinct from the X chromosome inactivation (XCI) in female somatic cells, MSCI does not depend on Xist RNA, a master regulator of XCI4,5. Instead, increasing evidence suggests that DNA damage response (DDR) factors act as both sensors and effectors in initiating MSCI6, 7, 8. Specifically, the serine/threonine protein kinase ATR, along with its activator TOPBP1, recognizes incompletely synapsed XY chromosomes9, 10, 11. ATR then catalyzes the phosphorylation of H2AX (γH2AX), which recruits MDC1. MDC1 subsequently facilitates the spreading of γH2AX across the XY chromatin, promoting further binding of ATR and TOPBP1, thereby establishing a positive feedback loop12. Mutations of DDR factors such as BRCA1, γH2AX, ATR, and MDC1 severely disrupt this process6, 12, 13. Following the initiation of MSCI by DDR factors, repressive epigenetic marks, such as H3K9me1/2/3, are acquired on the XY chromosomes during early PAC3, 14. However, the repressive histone modifications carried by the H3-H4 dimers, except for H3K9me1, are lost in the early-to-mid PACs coinciding with the replacement of histones H3.1/H3.2 with H3.3, until the recovery of H3K9me2/3 in diplotene spermatocytes (DIPs)15. Additionally, repressive histone modifications, H3K27 methylation and H2AK119ub, are also excluded from the XY body during this period16, 17. Therefore, additional yet-to-be-discovered regulatory mechanisms likely contribute to gene silencing during the MSCI establishment in PACs.

The nucleolus is the largest nuclear membrane-less organelle assembled through liquid-liquid phase separation (LLPS) and is responsible for ribosome-subunit biogenesis18. Increasing evidence indicates that nucleolus also plays critical roles in other biological processes including DNA repair, stress response, genome organization, and transcription regulation19, 20, 21, 22. Intriguingly, previous transmission electron microscopy imaging data showed that the canonical nucleolus is detached from nucleolar organizing regions (NORs), and nucleolar components migrate from autosomes to the XY body in PACs23, 24, where they are likely not engaged in rRNA synthesis25. A recent study further found that pre-ribosomal RNAs (pre-rRNAs) are also present in the XY body and may recruit DDR factors to DNA damage sites26. Nevertheless, the dynamics and underlying mechanisms of nucleolar migration and its potential functions in MSCI remain largely unknown. Here, using 3D structured illumination microscopy (3D-SIM), we systematically examined the dynamic distribution of multiple nucleolar components during meiotic prophase in mouse spermatogenesis. Using a panel of genetic mouse models, we found that SENP3, NPM1, and rRNA are recruited to the XY body downstream of DDR factors and are essential for MSCI establishment. Mechanistically, these nucleolar components promote phase separation that excludes RNA polymerase II (Pol II) from the XY body. These results provide insights into the non-canonical functions and mechanisms by which nucleolar components contribute to the transcriptional regulation of MSCI in meiosis.

Results
To systematically investigate nucleolar dynamics during spermatogenesis, we isolated spermatogonia and spermatocytes spanning LEP-to-ZYG, early-to-mid and mid-to-late PAC, and DIP stages by fluorescence-activated cell sorting (FACS) based on Hoechst 33342 staining (Supplementary Fig. 1A), as previously described27, 28. We then examined the three canonical nucleolar subcompartments fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC) of the nucleolus using established markers: nucleolar transcription factor 1 (UBF) for the FC29, fibrillarin (FBL) for the DFC30, and nucleophosmin 1 (NPM1) for the GC31 by 3D-SIM (Fig. 1A). We confirmed that nucleoli in spermatogonia and LEP-to-ZYG spermatocytes exhibited the canonical three-layered structure, with FC, DFC, and GC arranged from the interior to the periphery (Fig. 1A). This organization underwent pronounced rearrangement during PAC. Starting at early PAC stage, NPM1 (GC) gradually migrated to the XY body, which was identified as a DAPI-dense region localized at the nuclear periphery13, 32, and completed this migration by mid-to-late PAC (Fig. 1A). As a consequence, the nucleolus outside the XY body adopted a two-layered FC-DFC configuration (Fig. 1A). In mid-to-late PACs, FBL (DFC) also migrated toward the XY body but accumulated specifically at one corner, whereas UBF (FC) remained outside the XY body (Fig. 1A). Notably, UBF nuclear signal progressively diminished from early PAC and became undetectable by the DIP stage (Fig. 1B), indicating complete disassembly of the canonical nucleolus outside the XY body. Because UBF stimulates RNA polymerase I (Pol I) to transcribe ribosomal DNA (rDNA)33, we examined rRNA synthesis during this transition. Pol I-dependent transcription generates 47S pre-rRNA, which is processed into 45S pre-rRNA and subsequently cleaved into 32S and 34S intermediates, the precursors of 18S, 5.8S, and 28S rRNAs in mice (Fig. 1C, left)18. Consistent with nucleolar disassembly, qPCR revealed a significant decline in nascent pre-rRNA in DIPs, as measured using primers specific to 45S pre-rRNA (Fig. 1C, right).

A similar dynamic redistribution of nucleolar components was also observed by co-immunostaining with γH2AX, an XY body marker13 (Supplementary Fig. 1B-C). Cell stages were determined based on the stages of seminiferous tubule segments by LECTIN staining34, 35. Among additional GC-associated proteins, SENP3 and TEX1036, 37, showed distinct behaviors: SENP3 mirrored the migration pattern of NPM1, whereas TEX10 resembled FBL (Supplementary Fig. 1B-C), indicating highly regulated spatial positioning and migrating timing among nucleolar components during spermatogenesis. Furthermore, combined immunostaining and RNA-FISH targeting γH2AX and 28S rRNA sequence revealed that rRNA, like NPM1 and SENP3, associates with the XY body in PACs (Supplementary Fig. 1B-C), consistent with a recent report26. In contrast, UBF (FC), FBL (DFC), NPM1, and SENP3 (GC) in PAC oocytes maintained the canonical three-layer structure without migration (Supplementary Fig. 1D). This indicates that nucleolar disassembly and migration during PAC stage are male-specific.

Taken together, our data reveal a progressive disassembly of the nucleolus outside the XY body during PAC, transitioning from a canonical three-layered (FC-DFC-GC) structure to a two-layered (FC-DFC) organization, and ultimately to a state that only one layer (DFC) can be detected outside the XY body in the nucleus of DIPs. Concurrently, nucleolar GC (indicated by NPM1) and DFC (indicated by FBL) components migrate sequentially to the XY body (Fig. 1D).

We next examined the migration of nucleolar components to the XY body at higher temporal resolution. Based on the staging of seminiferous tubules, PACs were subdivided into 10 substages (stage I–X PAC)38. NPM1, SENP3, and rRNA exhibited highly similar migration dynamics. In stage I PACs, all three appeared as dispersed puncta outside the XY body. They began to accumulate at the XY body in stage II-III PACs and progressively expanded to cover the entire XY body from stage IV to stage X (Fig. 1E; Supplementary Fig. 2A). Upon entry into DIP, NPM1, SENP3, and rRNA further aggregated and partially covered the XY body, forming a cap-like structure surrounding γH2AX signals (Fig. 1E; Supplementary Fig. 2A). Using an antibody specifically recognizing pre-rRNAs containing the 5.8S rRNA sequence26, co-immunofluorescence analyses revealed that NPM1, SENP3, and rRNA were largely colocalized throughout the PAC and DIP stages (Supplementary Fig. 3A-C). In PACs, these components initially accumulated within the dense body, a subdomain of the XY body characterized by a single, circular, DAPI-negative structure39 (Supplementary Fig. 3B-C, white arrowheads). Within the dense body, their spatial distributions were partially distinct, with NPM1 adopting a ring-shaped distribution, whereas SENP3 and rRNA appeared as punctate signals (Supplementary Fig. 3B-C, white arrowheads). In contrast, FBL migrated to the XY body at later stages, beginning at stage VII PAC, and remained restricted to one corner of the XY body until DIP, without spreading across the whole XY body (Supplementary Fig. 2A). Notably, this spatial segregation was conserved across species. In human spermatocytes, FBL similarly localized to a single corner of the XY body (Supplementary Fig. 2B), whereas NPM1 progressively migrated to and covered the XY body in stage IV–X PACs (Supplementary Fig. 2C), indicating an evolutionarily conserved pattern of nucleolar component redistribution during MSCI.

To determine the roles of NPM1 and SENP3 in spermatogenesis, we deleted Npm1 or Senp3 in spermatocytes by crossing Npm1flox/flox or Senp3flox/flox mice with Stra8-Cre transgenic mice, which induced recombination starting from spermatogonia40 (refer to as Npm1SKO or Senp3SKO) (Supplementary Fig. 4A-B). Immunoblot analyses confirmed a marked reduction of NPM1 or SENP3 proteins in the whole testes of Npm1SKO or Senp3SKO mice (Supplementary Fig. 4C-D). Residual signals were likely derived from Sertoli and Leydig cells. Both Npm1SKO and Senp3SKO males developed to adulthood and appeared grossly normal. However, they were completely infertile as no pups were produced after mating with wild-type (WT) females for more than 9 months. Consistently, testes from Npm1SKO and Senp3SKO males were much smaller than those of WT controls (Fig. 2A; Supplementary Fig. 4E). Seminiferous tubule diameters were reduced by more than 50% (Fig. 2B; Supplementary Fig. 4F), and almost no mature spermatozoa were detected in the epididymal lumens of either mutant (Fig. 2C; Supplementary Fig. 4G). All spermatocytes in Npm1SKO testes and 96.4% of spermatocytes in Senp3SKO testes were arrested at stage V PAC (Fig. 2B; Supplementary Fig. 4F-H). The small fraction of Senp3SKO spermatocytes that progressed beyond stage V PAC ultimately died at the step 14 spermatid stage (Supplementary Fig. 4H). In sum, these results demonstrate that NPM1 and SENP3 are essential for normal spermatogenesis and male fertility.

We next investigated how Npm1 and Senp3 deletion affects nucleolar migration during spermatogenesis. Immunofluorescence analyses showed that in both Npm1SKO and Senp3SKO spermatocytes, FBL and TEX10 remained outside the XY body without migration (Supplementary Fig. 5A). In Senp3SKO mice, the localization of NPM1 and rRNA was indistinguishable from that in WT cells from the spermatogonia to stage I PAC (Fig. 2D-E). However, in most stage II-V Senp3SKO PACs, neither NPM1 nor rRNA migrates to the XY body (Fig. 2D-E). Instead, they were randomly distributed throughout the nucleus as small patchy fluorescence signals (Fig. 2D-E). This defect was even more pronounced in Npm1SKO mice, in which SENP3 and rRNA signals were largely undetectable in the nucleus from spermatogonia to PACs (Fig. 2D-E), indicating that the nuclear localization of SENP3 and rRNA depends on NPM1, which may further enable their aggregation and migration to the XY body.

To further explore the molecular relationships among NPM1, SENP3, and rRNA during spermatogenesis, we next examined their interactions. SENP3 has been shown to directly interact with NPM1 in a yeast two-hybrid system41. Consistently, co-immunoprecipitation (Co-IP) analyses in WT spermatocytes revealed a robust interaction between NPM1 and SENP3, which remained stable even after RNase A and DNase treatment (Supplementary Fig. 5B). In line with previous reports showing that SENP3 catalyzes NPM1 deSUMOylation in interphase nucleoli42, we observed a 3.5-fold increase in SUMOylated NPM1 in Senp3SKO PACs, while total NPM1 protein levels remained largely unchanged (Supplementary Fig. 5C). RNA immunoprecipitation (RIP)-qPCR using WT spermatocyte lysates further demonstrated that NPM1, but not SENP3, directly binds rRNA (Supplementary Fig. 5D). Notably, the rRNA-binding ability of NPM1 was significantly reduced in Senp3SKO spermatocytes (Supplementary Fig. 5D). Together, these results suggest that SENP3-mediated deSUMOylation of NPM1 facilitates its interaction with rRNA, thereby enabling proper nucleolar aggregation and migration during spermatogenesis.

To assess chromosomal defects caused by NPM1 or SENP3 deletion, we examined meiotic chromosomal synapsis by staining SYCP1, a marker of synapsed chromosomes, and SYCP3, a chromosome axis marker. In both Npm1SKO and Senp3SKO PACs, autosomes were fully synapsed, comparable to WT PACs (Supplementary Fig. 6A-B). As previously reported43, in WT PACs, XY chromosomes undergo nonhomologous synapsis beyond the PAR and exhibit a flexible bending configuration in early PACs. They subsequently separate from autosomes and synapse only at the PAR in mid PACs, and by late PAC, their axes elongated and were highly folded within the XY body (Supplementary Fig. 6A). In contrast, in Npm1SKO PACs, 18.65% of XY chromosomes failed to synapse (Supplementary Fig. 6B, row 2 and 6C), and 14.22% of X chromosomes exhibited self-synapsis (Supplementary Fig. 6B, row 3 and 6C). The remaining spermatocytes showed normal PAR synapsis (Supplementary Fig. 6B, row 1 and 6C). Similar defects were observed in Senp3SKO PACs, with 11.97% exhibiting XY asynapsis and 11.27% showing X chromosome self-synapsis (Supplementary Fig. 6B-C).

Even among Npm1SKO and Senp3SKO PACs with apparently normal PAR synapsis, the X chromosomes were markedly elongated compared with WT controls, as revealed by SYCP3-stained chromosomal axes (Supplementary Fig. 6A-B). To quantify this defect, we performed combined SYCP3 and CREST (a centromere marker) staining and measured the mean X-Y centromere distance in PACs with normal PAR synapsis, as previously described12, 44. The X-Y distance in Npm1SKO and Senp3SKO PACs was approximately 2-fold longer than that in WT PACs (Fig. 3A). Consistently, Hi-C analyses of FACS-purified early-to-mid PACs revealed reduced interactions perpendicular to the major interaction axes of the X chromosome in Senp3SKO cells, indicative of impaired hairpin-like folding of the X chromosome (Fig. 3B, black arrows). These results indicate that although autosomal synapsis is preserved, XY chromosome synapsis and condensation are compromised in the absence of NPM1 or SENP3.

To further examine XY body formation, we identified XY chromosomes by their lack of full-axis SYCP1 signals and analyzed DAPI patterns in PAC chromosome spreads. In WT PACs, XY chromosomes exhibited dense DAPI staining (Supplementary Fig. 6D), characteristic of heterochromatin formation13, 32. In contrast, the proportion of cells lacking this feature was significantly increased in Npm1SKO and Senp3SKO PACs (Supplementary Fig. 6D). Moreover, in WT PACs, γH2AX-marked XY chromosomes gradually compacted into a condensed heterochromatic sphere from stage I onward (Fig. 3C). By contrast, approximately 70% of Npm1SKO and Senp3SKO PACs displayed donut-like XY body structures, compared with ~10% in WT PACs) (Fig. 3C-D). 3D reconstruction revealed that these abnormal XY bodies adopt a hollow spherical structure (Fig. 3C) (all other immunostaining images were generated with maximum projection). Collectively, these data demonstrate that NPM1 and SENP3 are jointly required for proper XY chromosome synapsis, condensation, and XY body formation during spermatogenesis.

The defective XY body formation observed in Npm1SKO and Senp3SKO mice prompted us to investigate whether MSCI was also disrupted. Using an antibody against RPB1, the largest subunit of Pol II, we found that Pol II failed to be excluded and abnormally accumulated in the XY body of Npm1SKO and Senp3SKO PACs (Fig. 4A). To further evaluate transcriptional activity, we performed immunostaining for Pol II phosphorylated at serine (Ser2P) and serine 5 (Ser5P), which mark transcriptional elongation and promoter pausing, respectively45. In WT PACs, both Ser2P and Ser5P signals were distributed throughout the nucleus except the transcriptionally silenced XY chromosomes (Supplementary Fig. 7A-B). In contrast, Npm1SKO and Senp3SKO PACs exhibited strong Ser2P and Ser5P accumulation within the XY body, comparable to the levels on autosomes (Supplementary Fig. 7A-B). These results indicate aberrant transcriptional activation within the XY body.

To further explore this, we performed RNA-seq with FACS-purified early-to-mid PACs from WT, Npm1SKO, and Senp3SKO mice (Supplementary Fig. 8A)27. The purity of sorted populations was confirmed by co-immunostaining for γH2AX and H1T, a nuclear marker that emerges in mid-PAC and gradually intensifies as PACs mature (Supplementary Fig. 8B)46. Globally, 1,913 and 1,167 genes were upregulated and downregulated in Senp3SKO PACs, whereas Npm1SKO PACs exhibited 4,339 upregulated and 2,388 downregulated genes (Fig. 4B). Upregulated genes were significantly enriched for cell-cycle and transcription-related functions, whereas downregulated genes were primarily associated with sperm development processes, including spermatogenesis, cilium biogenesis and degradation, and fertilization (Supplementary Fig. 9A). Among XY-linked genes, approximately half were differentially expressed (DE) compared to WT PACs (370 in Senp3SKO and 469 in Npm1SKO PACs). Notably, the vast majority were upregulated (93.8% in Senp3SKO and 99.8% in Npm1SKO PACs) (Fig. 4B-C). This contrasts with the more balanced ratio of upregulated and downregulated autosomal genes (57.8% vs. 42.2% in Senp3SKO and 61.9% vs. 38.1% in Npm1SKO PACs) (Fig. 4B-C). Furthermore, 95.1% of upregulated XY-linked genes in Senp3SKO PACs were also upregulated in Npm1SKO PACs, while 70.5% of those in Npm1SKO PACs overlapped with Senp3SKO PACs (Fig. 4D). We and others previously reported that PACs undergo genome-wide dissolvement of conventional TADs and compartments A/B, while retaining refined local compartments that alternate between transcribing and non-transcribing regions on autosomes (Refined A/B), but not on X chromosomes47, 48, 49, 50, 51. Consistent with RNA-seq results, Hi-C analysis revealed that genomic regions enriched for upregulated genes exhibited prominent plaid patterns in Senp3SKO PACs compared with WT controls (Supplementary Fig. 9B, black and white arrows). These changes were not due to developmental arrest, as HIT and γH2AX staining confirm the early-to-mid PAC stage of spermatocytes in both control and Senp3SKO mice (Supplementary Fig. 8B), and insulation score analysis showed that TADs remain stronger in Senp3SKO PACs compared to WT ZYGs (Supplementary Fig. 9C). Taken together, these results suggest that NPM1 and SENP3 are essential for MSCI in PACs.

We next investigated whether rRNA is also required for XY body formation and MSCI by inhibiting Pol I-mediated transcription with BMH-21, which induces rapid Pol I degradation52. In control spermatocytes, the catalytic Pol I subunit RPA194 exhibited punctate nuclear signals from LEP to PAC and disappeared by DIP (Supplementary Fig. 10 A). Consistently, 45S pre-rRNA levels declined markedly in DIPs (Fig. 1C and 4E), indicating active rRNA transcription prior to nucleolar disassembly. Treatment with 10 μM BMH-21 for 3 h resulted in near-complete loss of nuclear Pol I signals (Supplementary Fig. 10A) and dramatically reduced 45S pre-rRNA levels (Fig. 4E). Similar results were obtained using CX-5461 (Fig. 4E; Supplementary Fig. 10A), an inhibitor that prevents recruitment of the Pol I initiation factor SL1 to rDNA53, 54. Co-immunostaining of MDC1 and rRNA, using an antibody against the 5.8S rRNA sequence, revealed that rRNA failed to distribute across the XY body following Pol I inhibition (Fig. 4F), indicating that rRNA localization to the XY body requires active rRNA transcription in PACs.

Next, we evaluated the impact on XY body formation and MSCI (Fig. 4G-H). In DMSO-treated controls, 89.23% of PACs displayed distinct DAPI intensity within MDC1-marked XY bodies (Fig. 4H, left), consistent with previous reports13, 32. In contrast, this proportion was significantly reduced following Pol I inhibition (Fig. 4H, right), indicating compromised XY body formation. Meanwhile, Pol II abnormally accumulated within the XY body (Fig. 4G), suggesting transcriptional reactivation. Taken together, these data establish a critical role for rRNA in XY body formation and MSCI.

Because XY chromosomes lack extensive homology, SPO11-induced DSBs cannot be repaired by homologous recombination at the onset of the PAC stage, leading to persistent DNA damage and recruitment of DDR factors that initiate MSCI10, 55. We therefore assessed whether nucleolar migration depends on DDR signaling. In chromosome spreads from Senp3SKO and Npm1SKO PACs, the localization and foci numbers of BRCA1, ATR, and MDC1 were comparable to WT controls (Supplementary Fig. 11A). Similarly, Pol I inhibition did not disrupt MDC1 recruitment to the XY body (Fig. 4F-G). These observations indicate that DDR factor recruitment is largely independent of NPM1, SENP3, and rRNA.

To determine whether DNA damage directly promotes NPM1 recruitment, we generated a Npm1EGFP knock-in mouse and induced DNA damage via laser irradiation. EGFP-NPM1 rapidly accumulated at damage sites in early-to-mid PACs and, to a lesser extent, in mid-to-late PACs (Fig. 5A-B). By contrast, no recruitment was observed in LEPs or ZYGs (Fig. 5A-B), indicating stage-specific responsiveness. Consistently, NPM1 accumulation on XY chromosomes was abolished in H2ax -/- and Mdc1-/- early and mid PACs (Supplementary Fig. 11B), demonstrating that DDR factors are required for NPM1 recruitment.

Furthermore, pharmacological inhibition of ATR signaling using AZD6738 abolished ATR signals and eliminated NPM1 localization to the XY body (Fig. 5C-E; Supplementary Fig. 12A). Consistent with previous reports that MSCI is reversible8, ATR inhibition led to Pol II accumulation and XY gene reactivation (Fig. 5F-G; Supplementary Fig. 12B). Following inhibitor removal, NPM1 rapidly reappeared on XY axes within 0.5 h and spread across the XY chromatin within 1 h, mirroring the recruitment pattern of ATR-dependent DDR factors, such as MDC1 (Fig. 5C-E), during MSCI initiation10, 12, 13. rRNA signals reappeared slightly later, initially localizing to the dense-body before expanding across the XY chromatin (Fig. 5E; Supplementary Fig. 12D). Collectively, these results suggest that NPM1 and rRNA recruitment to the XY body is DDR-dependent and reversible, indicating that NPM1, SENP3, and rRNA function downstream of DDR signaling during MSCI.

We next investigated the mechanism by which NPM1, SENP3, and rRNA regulate transcriptional silencing during MSCI. LLPS of NPM1 in the presence of rRNA has been proposed to drive multilayer nucleolar organization31. We therefore examined whether NPM1 undergoes LLPS within the XY body. Fluorescence recovery after photobleaching (FRAP) analysis in Npm1EGFP PACs showed rapid fluorescence recovery (∼10s) following photobleaching in both early and mid-to-late (stage IV-X) PACs (Fig. 6A-C). Live-cell imaging further revealed fusion behavior of EGFP-NPM1 condensates in the XY body (Fig. 6D; Supplementary Movie 1). These data suggest that NPM1 exhibits liquid-like properties in the XY body.

This raised an interesting possibility that phase-separated NPM1 may exclude the transcription machinery—another liquid-phase-separated condensate formed by transcription factors and related proteins56—from the XY chromosomes. We reconstituted transcription-initiating condensates in vitro using the C-terminal domain of Pol II (CTD), TATA-box binding protein (TBP), and total RNA as previously described57. Similar to in vivo observations58, recombinant CTD was incorporated into TBP condensates (Fig. 6E, row 1). However, when NPM1 and rRNA were added, a distinct multiphase condensate formed. TBP and Pol II CTD localized to the condensate periphery, whereas NPM1 and rRNA occupied the interior (Fig. 6E, row 2; Supplementary Fig. 13A). The condensates underwent fusion on contact (Supplementary Movie 2) and rapidly recovered after photobleaching (30 s to recover 80%) (Supplementary Fig. 13C). Over time, Pol II CTD was gradually released from the TBP condensates, with fluorescence intensity decreasing to ~15% of the initial level within 18 min (Supplementary Fig. 13A-B; Supplementary Movie 3). These results suggest that NPM1-rRNA condensates destabilize transcription initiation complexes. We next examined elongation condensates using phosphorylated CTD (pCTD) and the SPOC domain of PHF3 (SPOC)59. SPOC serves as a Pol II CTD reader that drives the LLPS of pCTD and facilitates Pol II invert into an elongation state59. Consistent with earlier findings, pCTD was incorporated into liquid-like SPOC condensates in vitro (Supplementary Fig. 13D-E). Addition of NPM1 and rRNA results in rapid exclusion of pCTD (Supplementary Fig. 13F). Most pCTD molecules were dispersed, while a small fraction remained associated with SPOC at the periphery of newly formed NPM1-SPOC condensates (Supplementary Fig. 13F). Fusion behavior and FRAP confirmed their liquid properties (Supplementary Fig. 13G-H). These results indicate that NPM1 phase separation interferes with both transcription initiation and elongation condensates in vitro.

Finally, we examined transcriptional output using an in vitro transcription system coupled with binary fluorescence quencher (BFQ)-based fluorescence resonance energy transfer (FRET) reporters (Fig. 6F)60. As expected, TBP enhanced transcription, reflected by reduced fluorescence signals (Fig. 6G, E6). When NPM1 and rRNA were added to the in vitro transcription system together, the fluorescence signals decreased much slower than that in the control (Fig. 6G, E4 vs E2), indicating a substantially lower transcriptional rate. Moreover, NPM1 and rRNA prevented TBP from promoting transcription (Fig. 6G, E7 vs E6). In comparison, the addition of NPM1 or rRNA alone only moderately inhibited the transcription (Fig. 6G, E3 or E5 vs E2). Taken together, these results demonstrate that NPM1 condensates exclude the transcription machinery from transcription-initiating condensates, leading to transcription inhibition in vitro.

We next asked whether NPM1 phase separation is essential for MSCI in vivo. Since hydrophobic residues are critical determinants of protein phase separation61, 62, we mutated Trp286 and Trp288 in mouse NPM1 (homologous to Trp288 and Trp290 in human NPM1) to alanine, generating the phase separation-defective mutant NPM1W286A,W288A, which lacks the hydrophobic pocket as previously reported (Supplementary Fig. 14A)63. Electrophoretic mobility shift assay (EMSA) showed that the RNA-binding capability of NPM1W286A,W288A was largely unaffected (Supplementary Fig. 14B). However, purified NPM1W286A,W288A proteins failed to form liquid droplets in vitro and instead remained soluble or formed irregular aggregates (Supplementary Fig. 14C). Consistently, overexpression of mCherry-tagged NPM1W286A,W288A in 293T cells resulted in a diffuse nuclear distribution, in contrast to the condensate-like pattern observed for mCherry-NPM1WT (Supplementary Fig. 14D). Together, these findings indicate that NPM1W286A,W288A loses its phase separation ability both in vivo and in vitro.

To examine the physiological consequence of impaired NPM1 phase separation, we generated Npm1W286A,W288A knock-in mice. Spermatocytes expressing only the mutant NPM1 protein were obtained from Npm1W286A,W288A/flox; Stra8Cre mice (hereafter referred to as Npm1CKI) by crossing Npm1flox/flox mice with Npm1W286A,W288A/+; Stra8Cre transgenic mice (Fig. 7A). Immunoblotting confirmed that NPM1W286A,W288A was expressed at levels comparable to WT NPM1 in Npm1CKI PACs (Supplementary Fig. 14E). Immunofluorescence analysis revealed that, unlike NPM1WT, NPM1W286A,W288A failed to accumulate in the MDC1-marked XY body and instead displayed a diffuse nuclear distribution in PACs (Fig. 7B), consistent with defective phase separation. Similar phenotypes were observed upon Pol I inhibition, where NPM1 no longer concentrated in the XY body but dispersed throughout the nucleoplasm (Supplementary Fig. 14F-G). Given that NPM1 phase separation depends on rRNA31, we speculated that Pol I inhibition disrupts pre-rRNA localization to the XY body, thereby preventing NPM1 condensation and recruitment. These results further support the notion that SENP3, NPM1, and rRNA function in a mutually dependent manner during MSCI. Taken together, these data demonstrate that NPM1 phase separation is essential for its localization to the XY body.

Phenotypically, Npm1CKI mice resembled germline-specific Npm1 knockout (Npm1SKO) mice, with males being sterile and all spermatocytes arrested at stage V PAC (Fig. 7C). To assess whether loss of NPM1 phase separation affects MSCI, we performed RNA-seq on FACS-purified early-to-mid PACs from control and Npm1CKI mice (Supplementary Fig. 15A). Cell purity was validated by co-immunostaining for H1T and γH2AX (Supplementary Fig. 15B). Compared with control PACs, about half of XY-linked genes (415) were differentially expressed in Npm1CKI PACs, with 99.8% (414/415) being significantly upregulated (Fig. 7D-E), indicating a severe defect in MSCI. Consistently, Pol II aberrantly accumulated within the XY body of Npm1CKI PACs (Fig. 7F). Moreover, the upregulated XY chromosomal genes in Npm1CKI PACs showed extensive overlap with those in Npm1SKO PACs (91.3% overlap for Npm1CKI and 80.8% for Npm1SKO) (Supplementary Fig. 14H), further supporting a shared underlying mechanism.

Finally, to determine whether NPM1 phase separation is required for excluding Pol II from the XY body in vivo, we overexpressed GFP tagged NPM1WT or phase separation-defective GFP-NPM1W286A,W288A proteins in Npm1SKO PACs through electroporation and quantified Pol II fluorescence intensity within the XY body. 18 h after electroporation, GFP-NPM1WT, but not GFP-NPM1W286A,W288A localized to the XY body, while the mutant protein remained diffusely distributed throughout the nucleus in both WT and Npm1SKO PACs (Fig. 7G, rows 3-4; Supplementary Fig. 14I). This is consistent with the diffuse distribution in the nucleus of Npm1CKI PACs (Fig. 7B), further confirming that NPM1 phase separation is essential for its localization in the XY body. Importantly, expression of GFP-NPM1WT, but not GFP-NPM1W286A,W288A, substantially reduced the aberrant accumulation of Pol II in the Npm1SKO XY body (Fig. 7G-H). Together, these results demonstrate that the phase separation ability of NPM1 is required for excluding Pol II from the XY body in PACs.

Methods
Study participants
Written informed consent was obtained from the three participants. This study was approved by the Institutional Ethical Committee of the School of Medicine, Shandong University with the approval number 2018-77, and by the Institutional Ethical Committee of Shanghai Jiaotong University School of Medicine, Renji Hospital with the approval number KY2021-173-B.

Mice
All mice were housed in the animal facility under SPF conditions following institutional guidelines and ethical regulations and fed with regular chow and water by the facility staff. The use of mice was approved by the Animal Ethics Committee of the School of Medicine, Shandong University. All animal care protocols were reviewed and approved by the Animal Use Committee of the School of Medicine, Shandong University. All animal experiments were performed according to approved institutional animal care and use committee (IACUC) protocols (#2018-77) of Shandong University. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

H2ax−/− mice were gifts from Linyu Lu (Zhejiang University).

Production of EGFP-tag Npm1 knockin mice
The EGFP-tag Npm1 knockin mouse model was created by Cyagen (Suzhou China). Briefly, the Kozak-EGFP-fusion protein linker was inserted upstream of the ATG start codon. To engineer the targeting vector, homology arms were generated by PCR using BAC clone RP23-323L8 as a template. The Neo cassette was flanked in the targeting vector by SDA (self-deletion anchor, Rox) sites. DTA was used for negative selection. C57BL/6N ES cells were used for gene targeting.

Production of CRISPR/Cas9-edited Senp3 conditional knockout mice
The Senp3 conditional knockout mouse model was created by GemPharmatech (China). Exon 3 to exon 7 was selected as the target site. Briefly, Cas9 mRNA and gRNA were transcribed in vitro. Cas9, sgRNA, and donor were microinjected into the fertilized eggs of C57BL/6J mice. Fertilized eggs were transplanted to obtain positive F0 mice that were confirmed by PCR and sequencing. A stable F1 generation mouse model was obtained by mating positive F0 generation mice with C57BL/6J mice. To obtain germline-specific knockout mice, we crossed Senp3flox/flox mice with Stra8-Cre mice69. The Stra8-Cre mice were a generous gift from Prof. Minghan Tong at the Chinese Academy of Sciences.

Production of CRISPR/Cas9-edited Npm1 conditional knockout mice
The Npm1 conditional knockout mouse model was created by Cyagen (Suzhou China). Exon 2 to exon 6 was selected as the target site. Briefly, to engineer the targeting vector, homologous arms, and the conditional knockout region were generated by PCR using BAC clone RP23-26M12 as the template. Cas9, gRNA, and the targeting vector were co-injected into fertilized eggs for conditional knockout mouse production. The pups were genotyped by PCR followed by sequencing analysis. To obtain germline-specific knockout mice, we crossed Npm1flox/flox mice to Stra8-Cre mice69.

Production of CRISPR/Cas9-edited Npm1(p.W286A and p.W288A) point mutation mice.
The Npm1 (p.W286A and p.W288A) point mutation mouse model was created by Cyagen (Suzhou China). Two mutation sites (p.W286A (TGG was mutated to GCA) and p.W288A (TGG was mutated to GCA)) were introduced by single-stranded DNA (ssDNA) synthesis. The gRNA sequence is TACAGRGCTATTCAAGATCTC-TGG. This gRNA worked together with the Cas9 protein to form a Ribonucleoprotein (RNP) complex. This RNP complex, along with the synthetic ssDNA, was then injected into fertilized eggs of the C57BNL/6J mouse strain. Upon the birth of the F0 generation mice, individuals carrying the p.W286A and p.W288A mutations were selected. These positive mice were then bred to establish a stable genetic line of the Npm1 point mutant mouse model. The ssDNA sequence is:
CCAAGTAAAGGGTGGAGTTCCATCCTTGAAAACTAAACAGGCATTTTAGACAACACATTCTTGGCAAGTG
AACCTGGACAACATTTATCAAACACAGTAGGGAAAGTTCTCACTTTGCATTATAAAAAGGACAGCCAGATA
TTAACTATTACAGAAATGAAGACAGAATATTTCAAACTGTTTAAACCCTTTTCTTAAAGAGATTTCCTTGCC
TGTGCGAGATCTTGAATAGCCTGTAAAGGAGGAAAAAAATGGTAAAGAAATGTTAACTTTATAGAATTAAA
CAAATTTCATTTCTTCAG.

Production of CRISPR/Cas9-edited Mdc1 knockout mice
The Mdc1 knockout mouse model was created by Cyagen (Suzhou China). Exon 5 to exon 7 was selected as the target site. Briefly, to engineer the targeting vector, homologous arms, and the knockout region were generated by PCR using BAC clone RP23-23H7 as the template. Cas9, gRNA, and the targeting vector were co-injected into fertilized eggs for knockout mouse production. The pups were genotyped by PCR followed by sequencing analysis.

Stages of Spermatogenesis
The seminiferous epithelium was defined into 12 stages based on the popular staging system reported by Hess et al. (2009)35, which is based on the Golgi status of round spermatids. We employed immunostaining for LECTIN as a marker for Golgi status34, 35 to determine the stage of seminiferous tubules. Within each stage, the cell types present in the seminiferous epithelium are well-defined, allowing us to precisely identify the stage of spermatogenic cells.

Spermatocyte spreading and immunofluorescence staining
Mouse testicular cells were prepared for surface spreading and subsequent immunofluorescence staining as previously described. Briefly, seminiferous tubules were incubated in hypotonic extraction buffer [30 mM tris (pH 8.2), 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 2.5 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF)] for 30 min at room temperature. Subsequently, a cell suspension was made in 100 mM sucrose and spread on slides with 1% PFA (pH 9.2) containing 0.15% Triton X-100. Slides were then placed in a humidified chamber for at least 1 h. Finally, slides were washed twice for 3 min in 0.4% Photoflo (Kodak) and air-dried at room temperature. Slides were either used for immunofluorescence staining immediately or stored at −80°C.

For immunofluorescence, slides were blocked for 30 min with 1× phosphate-buffered saline (PBS) containing 5% BSA. Slides were then incubated with primary antibodies overnight at 4℃ in a humidified chamber. Slides were then washed three times in 1× PBS. Secondary antibodies were applied for 1 h at room temperature in a humidified chamber. After secondary antibody incubation, three washes were performed in PBS and the slides were mounted with VECTASHIELD mounting medium (H-1000, Vector Laboratories). The primary and secondary antibodies used and their dilutions are shown in Supplementary Table 1.

Immunostaining and histological assays
Mice testes were fixed in 4% PFA overnight, dehydrated, embedded in paraffin, and sectioned at 5μm. After deparaffinization, rehydration, and antigen retrieval, tissue sections were permeabilized with 0.1% Triton X-100 in PBS, and incubated with primary antibodies overnight at 4° C. After this, Alexa Fluor 488, 594, or 647-conjugated secondary antibodies were added and incubated for 1 h at room temperature, with nuclei counterstained by DAPI (Vector Laboratories). Laser confocal scanning images were captured using an Andor Dragonfly spinning disc confocal microscope driven by Fusion Software. Projection images were then prepared using Bitplane Imaris (version8.1) software. The antibodies used are shown in Supplementary Table 1 in the Supplementary Information. For hematoxylin staining, testicular, epididymal, and ovarian tissues were fixed in Bouin’s buffer and embedded in paraffin, and 5 μm paraffin-embedded testicular or epididymal sections were sequentially deparaffinized, rehydrated, and stained with hematoxylin.

Human testicular biopsy samples were obtained from three adult patients (25–35 years old) diagnosed with obstructive azoospermia with normal spermatogenesis. Testicular tissues were collected for diagnostic purposes with informed consent from all participants and in accordance with institutional guidelines. Samples were fixed overnight in 4% PFA. Sectioning and immunofluorescence staining of human testis samples were performed using the same procedures as described above for mouse samples.

Structured Illumination Microscopy (SIM) slide preparation
After FACS, sorted spermatogenic cells were collected by centrifugation at 300×g and washed once with PBS. The cell pellet was gently resuspended in fixation buffer (1% PFA with 0.15% Triton X-100). Approximately 1×10⁵ cells were resuspended in 10 µL fixation buffer and incubated at room temperature for 5 min before being applied onto glass slides. The cell suspension was spread horizontally across the slide using the side of a 10 µL pipette tip to evenly distribute the cells. Slides were then air-dried and processed for downstream staining.

SIM procedure
All SIM experiments were performed on a DeltaVision OMX SR system (GE Healthcare) equipped with a 60 × /1.42 NA Plan Apo oil-immersion objective (Olympus) and four laser beams (405, 488, 568 and 642nm; 100mW). The microscope was routinely calibrated with a special image registration slide and algorithm provided by GE Healthcare. To obtain optimal images, immersion oil with a refractive index of 1.516 was used at 25 °C. SIM image stacks were captured with a z-distance of 0.125 µm and with 5 phases, 3 angles, and 15 raw images per plane. The raw data were reconstructed with channel-specific OTFs and a Wiener filter was set to an optimum value by using the softWoRx 6.5 package (GE Healthcare).

FISH coupled with immunofluorescent assays
Frozen testicular sections were used for RNA FISH analysis. RNA-FISH assays were performed according to the manufacturer’s protocol provided by the FISH Kit for frozen sections (GenePharma). Briefly, after fixation in 4% paraformaldehyde, tissues were cryoprotected through a sucrose gradient, embedded in OCT, and sectioned at a thickness of 5 µm. Frozen sections were rehydrated by adding buffer B to each section and incubating at room temperature for 15 min. Sections were incubated with protease K at 37℃ for 20 min. Sections were blocked by adding a blocking buffer at 37°C for 30 min and then denatured at 78°C for 8 min. After alcohol gradient dehydration, hybridization was carried out with a FISH probe in a humidified chamber at 37°C in the dark overnight. RiboBio designed and synthesized the 5.8S and 28S FISH probes. Sections were washed three times with Wash Buffer I (4× SSC with 0.1% Tween-20, Sigma) and then with Wash Buffer II (2× SSC, Sigma) and Wash Buffer III (1× SSC, Sigma) at 42°C in the dark for 10 min. Following RNA-FISH, γH2AX immunostaining was carried out on the same slides. Sections were incubated with γH2AX primary antibody (Supplementary Table 1) for 2 h at room temperature, washed three times with PBST, and then incubated with secondary antibody (Supplementary Table 1) for 1 h. After three additional washes, slides were mounted with VECTASHIELD (H-1000, Vector Laboratories). Images were acquired using an Andor Dragonfly spinning-disk confocal microscope.

Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting assays were performed with standard protocols. In brief, mouse tissues or cells were homogenized in lysis buffer [50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 20mM NaF, 20 mM β-glycerophosphate, 1% NP-40, 1 mM EDTA, 6 mM EGTA, 1 mM DTT, and proteinase inhibitor cocktail (Roche)]. Primary antibody-coupled Protein A/G beads were added to the precleared tissue or cell lysates and incubated overnight at 4°C. After washing with washing buffer [50 mM Tris-HCl (pH 7.4), 350 mM NaCl, 20 mM NaF, 20 mM β-glycerophosphate, 1% NP-40, 1 mM EDTA, 6 mM EGTA, 1 mM DTT], immunoprecipitation pellets or tissue/cell extracts were diluted in SDS-loading buffer and then analyzed with standard SDS-PAGE and immunoblotting procedures. Western blotting images were captured by a Tanon-5200 Chemiluminescent Imaging System (Tanon). The antibodies used are shown in Supplementary Table 1 in the Supplementary Information.

Isolation of spermatogenic cells
Testes were collected in PBS and placed on ice. After removal of the tunica albuginea, the testes were incubated in 5 mL of PBS containing 120 U/mL of collagenase type I at 32°C with gentle agitation for 5 min. The dispersed seminiferous tubules were further digested with 5 mL of 0.25% trypsin and 0.1 mL of DNase I (5 mg/mL) by pipetting gently several times at 32°C for 8 min, and the digestion was terminated by adding 0.5 mL of fetal bovine serum (FBS) to inactivate trypsin. Following the two-step enzymatic digestion, the dissociated testicular cell suspension was filtered through a PBS pre-wetted cellular filter with pore size of 70 μm. The cell suspension was centrifuged at 100g for 5 min at 4°C, and the supernatant was carefully removed from the pellet. The cells in the pellet were resuspended at a concentration of 1×106 cells/mL in DMEM with Hoechst 33342 (3 mg/mL) and 5 μl DNase I. For FACS, the cell suspension was rotated for 20 min at 32°C in the oven at 10 rpm (the minimum speed) then centrifuged at 100g for 5 min at 4°C and resuspended in 1–2 mL DMEM for sorting. Cell populations were collected into a 1.5ml tube containing 400μl of 100% FBS based on their fluorescent label with Hoechst 33342 staining using FACS.

ATR inhibitor treatment
ATR inhibition and recovery were performed as described recently8. Briefly, following FACS, spermatocytes were cultured in a culture medium70 containing DMSO or ATR inhibitor (AZD6738, Selleck Chemicals, 5μM). After 24 h culture, the viability of spermatocytes was measured using a Countess 3 FL Cell counter (Invitrogen). For recovery experiments, both control and AZD6738-treated cells were collected after 24 h incubation by centrifugation at 100g for 10 min at room temperature (RT). Next, the cells were washed twice by suspending in the control culture media containing DMSO and centrifuging at 100g for 10 min at RT. The cell pellets that were obtained were then resuspended in a culture medium containing DMSO, and the cells were incubated for an indicated time at 32 °C with 5% CO2 air.

Pol I inhibitor treatment
Similar to ATR inhibitor treatment, spermatocytes collected were treated with 10μM BMH-21(Selleckchem) or 10μM CX-5461(Selleckchem) for 3h. The cells were incubated at 32 °C with 5% CO2 air. After 3h incubation, spermatocytes were collected for meiotic chromosome spreads.

sisHi-C library generation and sequencing
The sisHi-C library generation was performed as described previously71. Briefly, WT and Senp3SKO spermatocytes were fixed with 1% formaldehyde at room temperature (RT) for 10 min. Formaldehyde was quenched with glycine for 10 min at RT. Cells were washed with PBS and then lysed in 50 µl lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 0.1 mM EDTA, 0.5% NP-40, and proteinase inhibitor) on ice for 30–50 min. After centrifuging at 3500 rpm at 4°C for 5 min, the supernatant was discarded with a pipette. Chromatin was solubilized in 0.5% SDS and incubated at 62°C for 10 min. SDS was quenched with 10% Triton X-100 at 37°C for 15 min. Then the nuclei were digested with 50U MboI at 37°C overnight with rotation. MboI was then inactivated at 62°C for 20 min. To add the biotin label to the DNA, dCTP, dGTP, dTTP, biotin-14-dATP, and Klenow were added to the solution and the reaction was carried out at 37°C for 1.5 h with rotation. The fragments were ligated at RT for 4–6 h with rotation. This was followed by reversal of crosslinking and DNA purification. DNA was sheared to 300–500 bp with a Covaris M220. The biotin-labeled DNA was then pulled down with 10 µl Dynabeads MyOne Streptavidin T1. Sequencing library preparation was performed on beads, including end-repair, dATP tailing, and adaptor ligation. DNA was eluted twice by adding 20 µl water to the tube and incubating at 66°C for 20 min. A total of 9–12 cycles of PCR amplification were performed with KAPA HiFi HotStart ReadyMix. Finally, size selection was done with AMPure XP beads and fragments ranging from 200 bp to 1000 bp were selected. All of the libraries were sequenced on an Illumina NovaSeq according to the manufacturer’s instructions.

Hi-C data processing and analysis
The paired end raw reads of Hi-C libraries were aligned, processed and iteratively corrected using HiC-Pro (version 2.11.1) as described72. Briefly, sequencing reads were first independently aligned to the mouse reference genome (mm9) using the bowtie2 end-to-end algorithm and “-very-sensitive option”. To rescue the chimeric fragments spanning the ligation junction, the ligation site was detected and the 5’ fraction of the reads were aligned back to the reference genome. Unmapped reads, multiple mapped reads and singletons were then discarded. Pairs of aligned reads were then assigned to MboI restriction fragments. Read pairs from uncut DNA, self-circle ligation and PCR artifacts were filtered out and the valid read pairs involving two different restriction fragments were used to build the contact matrix. Valid read pairs were then binned at a specific resolution by dividing the genome into bins of equal size. Next, the binned interaction matrices were normalized using the iterative correction method72, 73, to correct the biases such as GC content, mappability and effective fragment length in the Hi-C data.

To compare the average TAD boundary strengths between different stages or genotypes, the insulation scores were calculated in 50-Kb resolution and 1-Mb window with the “diamond-insulation” function of cooltools (version 0.3.2)74. TAD boundaries previously identified in mESCs75 were used as representatives of normal TADs.

For comparison of Hi-C data between different stages, WT, and mutant, the same numbers of randomly sampled valid pairs were used to avoid potential bias caused by sequencing depths.

Local PC1 analysis
To determine the localizations of refined compartment A/B, local PC1 values were called as previously described48, 76. Briefly, the local interactions (chr12: 58-74Mb and chrX: 35-50Mb) were used instead of those from the whole chromosomes. Principal component analysis was performed on the correlation matrices generated from the observed/expected matrices. The directions of local PC1 values were adjusted according to the gene density.

RNA-seq library preparation and sequencing
Cells and embryos were lysed in lysis buffer (0.2% v/v) Triton X-100 and 2 U/μl RNase inhibitor). The polyadenylated mRNAs were captured by the oligo-dT primers and subjected to the Smart-seq2 protocol as described previously77.

Analysis of RNA-seq datasets
All RNA-seq data were mapped to the mm9 genome by HISAT2 (version 2.2.0)78. The gene expression level was calculated using StringTie (version 2.1.1) based on the annotation mm9 refFlat database from the UCSC genome browser79. The normalized differential expression analyses were performed by DESeq280. Differentially expressed genes were called if the associated adjusted p-value was less than 0.05. To compare the fold changes of autosomal and X-linked genes, only genes with fragments per kilobase of transcript per million mapped exonic reads (FPKM) >0.1 were considered.

RNA immunoprecipitation (RIP)
The RIP assay was performed using the RNA immunoprecipitation kit (Geneseed Biotech, China# P0101) according to the manufacturer's instructions. A total of 1×107 tetraploid spermatocytes were lysed with Buffer A for 30 min on ice, and 3 μg of antibodies were used for immunoprecipitation in each RIP assay. cDNA was synthesized with the PrimeScript™ RT reagent Kit (Takara, RR037A) and then analyzed by qRT-PCR using SYBR® Premix Ex Taq™ (Takara, RR420A). The enrichment was calculated relative to the percentage of input. The qPCR primers for 5.8S and 28S rRNA are shown in Supplementary Table 2 in the Supporting Information. The antibodies used are shown in Supplementary Table 1 in the Supporting Information.

Laser micro irradiation
Laser micro irradiation was carried out on a laser scanning confocal microscope (Nikon A1R HD25). A frame of image was captured before laser micro irradiation for normalization. Spermatocytes were irradiated using a 355nm laser at 50% laser power. Images were taken at constant intervals over the recovery period. The time-lapse images were analyzed using NIS-Element AR5.2 software, and the fluorescence intensity change was normalized to the intensity before laser micro irradiation.

Protein expression and purification
The pET28b-His-TEV-TBP and pET-28a-CTD52-eGFP constructs were generous gifts from Xiaohua Shen at Tsinghua University. Wild-type cDNA of NPM1 was subcloned into pTac vector with His N-terminal mCherry/ His N-terminal BFP/His tag. The Tac promoter was replaced with the CMV promoter to construct the pCMV-His N-terminal mCherry-NPM1 vector. pCAG-GFP-NPM1 vector was constructed by insertion of the coding region of NPM1 into the pCAG-GFP vector. NPM1 (p.W286A and p.W288A) point mutation vectors were generated using the Site-Directed Mutagenesis Kit (New England Biolabs). PHF3 SPOC was additionally cloned into pET Duet 6xHis-TEV-mCherry between SacI and NotI. All constructs were verified by DNA sequencing.

Recombinant proteins were expressed in E. coli strain BL21. Cultures were grown in Luria broth medium at 37°C to an OD of 0.6. After induction with 0.4 mM isopropyl-β-D-thiogalactopyranoside, the cells were grown at 16°C overnight, harvested by centrifugation at 4°C, and then lysed with an Ultrasonic Cell Disruptor (Scientz) in binding buffer (30 mM Tris-HCl, pH 8.0, 300mM NaCl, 5 mM imidazole, and 1 mM phenylmetha-nesulfonyl fluoride (PMSF)). The bacterial lysate was clarified by centrifugation at 20,000g for 40min. The supernatant with recombinant protein was loaded onto a HisTrap HP column (Smart-Lifesciences). After extensive washing with washing buffer (30 mM Tris-HCl, pH 8.0, 300mM NaCl, 50 mM imidazole), the target protein was eluted in an elution buffer (30 mM Tris-HCl, pH 8.0, 300mM Nacl, 500 mM imidazole, and 1 mM PMSF). Proteins were further loaded onto a SpinDesalt Column (Smart-Lifesciences). The target protein was eluted and dialyzed in a buffer with a target concentration of NaCl.

Protein fluorescent labeling
His-TBP protein was labeled by Alexa Fluor 594 NHS Ester (Succinimidyl Ester) (Invitrogen#A20004) with a molar ratio of 1:10 (dye: protein) at room temperature for 1 h. The free dye was removed using a SpinDesalt Column (Smart-Lifesciences#SEC02301) before the in vitro phase-separation assay.

In vitro transcription and Cy5 labeling of rRNA
To generate Cy5-labeled rRNA, the 5.8S rRNA sequence was cloned into the pET28a vector, and linear DNA templates were prepared by single-enzyme digestion with XhoI (Invitrogen, FD0694) followed by gel purification (TIANGEN, DP219). In vitro transcription was performed using the HyperScrib T7 High Yield Cy5 RNA Labeling Kit Plus (APEX-Bio, K1404) according to the manufacturer’s protocol, with reactions incubated at 37 °C for 16 h. After transcription, Cy5-rRNA was purified using a standard TRIzol-chloroform extraction with Phase-Lock tubes (TIANGEN), followed by isopropanol precipitation at −20 °C and ethanol washing. The final RNA pellet was air-dried briefly and resuspended in RNase-free water to obtain purified Cy5-labeled rRNA.

Condensate formation and live cell imaging
The droplet-formation assay was carried out in buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, and 5 mM DTT, with phase-separation components mixed at the concentrations indicated in the manuscript. Live-cell imaging was performed on an Andor Dragonfly spinning-disk confocal microscope with a 40×oil-immersion objective. Excitation lasers used were: 405 nm for BFP-NPM1, 488 nm for GFP-CTD, 561 nm for 594 NHS-Ester–labeled TBP, and 637 nm for Cy5-labeled rRNA.

Cell culture and transfections
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Transfections were performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. All DNA plasmids were cleared of endotoxin. For live-cell imaging experiment, 1.5 mg of plasmids of mCherry-tagged NPM1 or mutants were transfected in a 35-mm dish with a glass-like polymer bottom (Cellvis, D35-20-1.5P). Images were captured using an Andor Dragonfly spinning disc confocal microscope driven by Fusion Software.

Electrophoretic mobility shift assay
WT or mutant NPM1 proteins were expressed and purified as described above. rRNA (BioWorld) and WT or mutant NPM1 protein were mixed at the indicated concentrations in the binding buffer containing 100 mM NaCl and 20 mM Tris-HCl, pH 7.5, and incubated on ice for 20 min. In the meantime, 1% agarose gel (BIOWEST) containing 0.5× TBE buffer (45 mM Tris-Borate, 1 mM EDTA) supplied with 1× SYBR Gold (ThermoFisher, catalog #S11494) was prepared. The incubated mixture was then subjected to the gel and run in 0.5× TBE buffer at 5V/cm for ~40 min. After the electrophoresis, the gel was scanned using a multi-automatic fluorescence image analysis system (Tanon 5200 Multi). The gray intensity of free and NPM1 or NPM1W286A/W288A-bound rRNA was measured with ImageJ, and GraphPad Prism 9 software was used to fit the binding curve of each reaction with a nonlinear regression method based on the Hill equation.

In vitro kinase assay
For reactions containing recombinant kinase complexes CDK9-CycT1 (P-TEFb) were purchased from Millipore. The recombinant GFP- CTD52 and P-TEFb were mixed 1:1 at 30 °C in a 10 µl reaction that also contained 50 mM HEPES pH 7.3, 50 mM NaCl, 1 mM DTT, 10 mM MgCl2, and 0.1 mM ATP for 6 h to ensure saturated phosphorylation81.

In vitro phase-separation assay
The phase-separation assay was performed on a confocal dish (Nest). The purified protein was assembled by diluting the protein from a high-salt storage buffer to a varying concentration buffer containing 20 mM HEPES, pH 7.5, 100 to 250 mM NaCl, ± 2.5% PEG at the indicated protein concentration. For the co-phase separation assay of NPM1 and rRNA, rRNA was purchased from BioWorld (11020001-4). The NPM1 protein and RNA were then mixed gently at the indicated concentrations and diluted from a high-salt storage buffer to a physiological buffer containing 20 mM HEPES, pH 7.5, and 150 mM NaCl. After incubation for 30 min, all images were obtained with an Andor Dragonfly spinning disc confocal microscope driven by Fusion Software. For the co-phase separation assay of TBP, GFP-CTD52, total RNA, rRNA and NPM1, total RNA was purified with TRIzol (Invitrogen) according to the manufacturer's instructions. The two mixtures, one containing TBP, GFP-CTD52, and total RNA, and the other containing NPM1 and rRNA, were separately incubated and then mixed. Observations were made immediately afterward. The experimental procedure for in vitro phase separation of SPOC, pCTD52, rRNA, and NPM1 was performed similarly.

FRAP
All FRAP experiments in spermatocytes were performed on a laser scanning confocal microscope (Nikon A1R HD25). A frame of image was captured before photobleaching for normalization. 488 nm laser (5% laser intensity) was used for EGFP tag fusion NPM1 protein in the Npm1EGFP mouse model, and images were taken at constant intervals over the recovery period. The time-lapse images were analyzed using NIS-Element AR5.2 software, and the fluorescence intensity change during the recovery was normalized to the intensity before photobleaching.

All in vitro FRAP experiments of recombinant protein condensates were performed on an Andor Dragonfly spinning disc confocal microscope through a Micropoint device. A frame of image was captured before photobleaching for normalization. Protein condensates were bleached for 1s with 405 nm laser (50% laser intensity) for BFP tag fusion NPM1 protein, 594nm laser (50% laser intensity) for mCherry tag fusion SPOC protein and images were taken at a constant interval over the recovery period. The time-lapse images were analyzed using Andor iQ software, and the fluorescence intensity change during the recovery was normalized to the intensity before photobleaching.

In vitro transcription
For in vitro transcription assays, 1 unit E. coli RNA polymerase (NEB), 250nM of the MBa and MBb probes, 50 nM DNA template and indicated proteins were mixed in transcription buffer (20 mM Tris pH 7.5, 30 mM MgCl2, 2.5 mM DTT and 5% glycerol) and incubated for 30 min. RNA synthesis was started by addition of 250 μM NTP mixture and immediately placed in an RT-PCR thermocycler for fluorescence detection at a constant temperature of 37 ℃.

DNA sequence:
ctgaaatgagctgttgacaattaatcatcggctcgtataatgtgtgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaac
caccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcactagcataaccccttggggcctctaaacgggtcttgaggggttttttgc
tg
MBa: 5’ FAM- CTGTTTGATGGTGGTTAACGGCG
MBb: TTGCCCCAGCAGGCGAAAATC- 3’ BHQ1

Spermatocytes electrical transfection
Plasmid DNA preparation for electrical transfection: GFP-Npm1WT or GFP-Npm1W286A/W288A plasmid DNAs (CAG promoter) were extracted from E. coli DH5a by alkaline lysis (MAXI-KIT; QIAGEN) and solubilized in HBS buffer (2% HEPES, 0.8% NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 0.1% glucose). Dilute plasmid DNA with HBS buffer to a final concentration of 1 μg/μl.

Pre-mix cells and plasmid DNA, ensuring each 100 μl aliquot contains 1×106 cells (90μl, dilute with Opti-MEM) and 10 μg (10μl) of DNA. Add preheated spermatogenic cell culture medium to culture plates. Label the electroporation cup and add DNA/cell mixture to the electroporation cup. Adjust the electroporator settings according to the preliminary experiment guidelines as shown in Table 1, aiming to identify optimal conditions. In this study, spermatocyte electroporation follows the 2nd condition.

Before placing the electroporation cup in the electroporator, gently tap it to eliminate any bubbles. Measure and record the resistance value by pressing the "Ω" button, ensuring it falls within the 30-55Ω range. Initiate the electroporation program by clicking the "Start" button. After electroporation, carefully remove the cup and use a dedicated pipette to transfer approximately 200-300μl of medium from the culture plate into the cup. Gently pipette to disperse any white cell clumps, then transfer the entire cell suspension back to its original culture plate. If needed, rinse the cup with an additional medium to ensure complete cell transfer. It is crucial to immediately transfer the cell suspension from the electroporation cup to a culture dish for cultivation. Avoid waiting until all cups have completed electroporation to transfer the cells, as delays can significantly reduce cell viability. Fluorescence expression typically begins 6 hours post-electroporation, with a recommended sampling time for spermatocytes at 24 hours.

Statistics & Reproducibility
The data used in this study were presented as mean ± standard error of the mean (SEM) in triplicate experiments unless otherwise stated. The statistical significance of the differences between the mean values for the different genotypes was measured by Student’s t-test with a unpaired, 2-tailed distribution. Representative pictures for microscopy imaging and western blotting were obtained from three independent experiments. For the statistical significance and sample size of all graphs, please see figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "First, I would like to commend the authors for their massive effort and the tremendous improvements made to the manuscript.\nThe authors have thoroughly addressed all of my original comments. I am also pleased to learn that my initial feedback\ncontributed to revisions in their mechanistic model, which now makes sense. This is truly a remarkable study and will mark a\nsignificant milestone in our understanding of MSCI and the nucleolus.\nGiven the novelty and significance of their findings, I am pleased to support publication. While I understand that the review\nrequest came from Nature Communications, I would also support publication in the original journal, [editorial note: journal\nname redacted]. I have no major concerns, only a few minor suggestions, which can be addressed without additional\nexperimentation.\nMinor Comments:\n1. Line 27: H3K27 methylation and H2AK119ub do not accumulate on XY chromosomes during male meiosis but rather are\nexcluded from XY. Please correct this statement.\n2. Line 62: The method used to prepare slides that preserve 3D nuclear structure after FACS cell isolation is not described.\nPlease include this information in the Methods section.\n3. The authors appear to have used three different methods for microscopy analysis of male meiosis (FACS-isolated cells,\ntissue sections, and chromosome spreads), but it is not clear which method was used for each image. Please clarify this in\nthe figure legends.\n4. The method for combining FISH and immunostaining is not described. Please include this information in the Methods\nsection.\n5. The live cell imaging method is not described. Specifically, how was CTD visualized in Fig. 6E? Was TBP detected using\nGFP? Please clarify.\n6. Line 120: This description does not match the negative Pearson correlation values shown in Fig. 1F. Could you revisit\nthis?\n7. Line 154: I do not observe any cytoplasmic signals for SENP3. Please review or clarify.\n8. Line 207: The donut-like signals are intriguing but could be a result of antibody penetration. Please provide more details\non the slide preparation methods for this data.\n9. I am unclear on how the right panel of Fig. 4F was quantified. Could you please explain the quantification method used in\nthe figure legend?\n10. Line 507: Please specify the \"diplotene stage\" for clarity.\n11. The model in Fig. 7I does not reflect DDR-dependent initiation of MSCI. I suggest revising it to more clearly illustrate the\ntwo steps: one involving DDR and the other nucleolar migration.\n12. For Fig. 6C, please include the number of independent experiments in the figure legend.", "reviewer_2": "The revised article by Li, et al., is improved relative to the original submission through addition of new data, revision to tone\ndown data interpretations and the addition of quantitation of the many fluorescence microscopy images. While improved,\nseveral issues need to be addressed before the manuscript could be considered for publication. The reviewer provides\nevaluations of the authors’ point-by-point responses to their original comments, noting experiments that require further\nattention. With appropriate revision, the manuscript will be of broad interest to the biomolecular condensates community as\nwell as scientists in the developmental biology field.\nPage 29 of rebuttal, general responses to Reviewer 2. The revisions to figures to include quantitation and statistics are\nacceptable.\nComment 1. See Comment 4, below.\nPage 5. “These results demonstrate that the LLPS ability of NPM1 is required for excluding Pol II from the XY body in PACs.”\nSuggest changing “demonstrate” to “support” to reflect the correlative nature of the data.\nComment 2. The revision is acceptable.\nComment 3. The revisions are acceptable.\nComment 4. “Additionally, live cell images showed that EGFP-NPM1 exhibited fusion behavior in the XY body. These data\nindicate that NPM1 exhibits liquid-like features and undergoes LLPS in the XY body.”\nThe still images in Fig. R1 B are suggestive of fusion by the NPM1 condensates but are not definitive. Perhaps a video\nwould be more convincing. The key events to capture are the rounding of an irregularly shaped condensate after fusion. The\nrounding-up is evidence of surface tension, a property of fluids. If the authors have access, they could possibly use lattice\nlight sheet microscopy to capture fusion events with higher resolution with the added benefits of reduced light load (e.g.,\nbleaching).\nFig. R1, C & D. While the FRAP data in panel C show the uniform recovery of fluorescence in a round condensate, the data\nin panel D appear grainy, suggesting that the material properties of the XY body have changed. The authors need to\naddress this apparent difference. Also, scale bars are missing from these two panels.\nSome of the data in Fig. R2, A-C are puzzling, and the authors comments about them require revision. The folded C-terminal\ndomain of NPM1 binds nucleic acids, including rRNA. Grummitt, et al., showed that the W286A, W288A mutations cause\nthis domain to unfold, abrogating interactions with rRNA and eliminating the localization of NPM1 in the nucleolus. Deletion\nof the C domain was additionally shown by Mitrea, et al. (eLife, 2016), to abrogate nucleolar localization of NPM1. These\neffects are due to loss of rRNA binding, not due to loss of hydrophobic interactions associated with phase separation. The C\ndomain unfolds with these mutations due to loss of hydrophobic interactions in the core of the domain, destabilizing the\nfolded structure. The authors need to revise their statements in this regard.\nGiven this, it is puzzling that both WT and WW mutant NPM1 bind rRNA in Fig. R2 B. What are these RNA species and why\nis it that the mutant binds as well as WT? Is the assay probing only non-specific interactions? Mitrea, et al., also showed that\nthe C domain is required for binding rRNA in vitro (Fig. 10B).\nPage 3. “Yet, the purified NPM1W286A,W288A proteins showed either a soluble state or fiber-like irregular aggregates in\nvitro (Fig. R2C).”\nThe objects in Fig. R3 C labeled “aggregates” do not appear to have fibrillar features and should not be referred to as such.\nPage 3. “These data strongly support the notion that the regulation of MSCI by NPM1 requires its phase separation ability.”\nSuggested revision: “These data strongly support the notion that the regulation of MSCI by NPM1 requires binding to and\nphase separation with rRNA.”\nThe data in Fig. R6 are acceptable.\nPage 32. “After 24 h treatment, the inhibitor was removed, and spermatocytes were collected at various time points for\nchromosomal spreads to monitor NPM1 recruitment. By 0.5 h inhibitor post-removal, NPM1 was first recruited to the XY\nchromosome axes, inferred from the distribution of NPM1 and SYCP3 signals. By 1 h, it had spread to XY chromatin. By 6 h,\nthe NPM1 signal recovered to approximately 60% of the level in DMSO treated controls (Fig. R29C-D). These results\nsuggest that NPM1 recruitment to the XY chromosomes follows and mirrors the dynamics of DDR factors, such as MDC1\n(Fig. R29C), which initially localize to the XY chromosomal axes and subsequently spread to chromatin loops (Turner,\n2015).”\nFig. R29 C. The recovery of MDC1 and NPM1 in this sequence of images requires quantitation. Also, the authors might also\nquantify the recovery of rRNA.\nComment 5. Fig. R30 A. The authors should also show imaging of rRNA as the condensates change over time.\n“In addition, the condensates underwent fusion on contact and recovered rapidly after photobleaching (10 s to recover 80%)\n(Fig. R30A, C).”\nFusion events are not evident in these images. The authors need to present images that clearly indicate condensate fusion\nevents.\nThe authors arguments regarding the incompatibility of TBP and CTD with NPM1/rRNA would be strengthened by\npresentation of quantitative analyses of the changes that occur in these condensates. Also, the authors should illustrate the\nstarting point of the experiment and the entire time sequence.\nComments 6 & 7. The revision is acceptable.\nComment 8. The new data with RNA Pol I inhibitors strengthens the manuscript.", "reviewer_3": "The authors have addressed my main concerns. I believe the study represents a valuable contribution to the understanding\nof nucleolar biology during mammalian spermatogenesis and recommend it for publication."}, "author_response": "GENERAL RESPONSES\nWe are delighted that reviewers are now generally supportive of our revised paper for publication. Here we\nsummarize our revisions of the manuscript to address the reviewers' remaining questions. Note that we refer\nto the figures and videos in the manuscript using \"Figure 1, 2, 3,\" etc., and \"Video 1, 2, 3,\" etc., and the figures\nand videos in this letter are referred to as \"Figure R1, R2, R3,\" etc., and \"Video R1, R2, R3,\" etc.\nPOINT-BY-POINT RESPONSES\nReviewer #1:\nRemarks to the Author:\nFirst, I would like to commend the authors for their massive effort and the tremendous improvements made to\nthe manuscript. The authors have thoroughly addressed all of my original comments. I am also pleased to\nlearn that my initial feedback contributed to revisions in their mechanistic model, which now makes sense.\nThis is truly a remarkable study and will mark a significant milestone in our understanding of MSCI and the\nnucleolus.\nGiven the novelty and significance of their findings, I am pleased to support publication. While I understand\nthat the review request came from Nature Communications, I would also support publication in the original\njournal, [editorial note: journal name redacted]. I have no major concerns, only a few minor suggestions, whi\nch can be addressed without additional experimentation.\nResponses: We sincerely appreciate the reviewer's encouraging comments. We are grateful that the reviewer\nrecognizes the substantial revisions and mechanistic insights incorporated into the revised manuscript, as well\nas the significance of our findings. We also truly appreciate the reviewer's insightful feedback during the initial\nsubmission, which greatly strengthened the study.\nFollowing the reviewer's suggestions, we have carefully revised the manuscript to address all remaining points.\nMinor Comments:\n1. Line 27: H3K27 methylation and H2AK119ub do not accumulate on XY chromosomes during male meiosis\nbut rather are excluded from XY. Please correct this statement.\nResponses: We thank the reviewer for pointing out this inaccuracy. We have now corrected the statement to\naccurately reflect that H3K27 methylation and H2AK119ub are excluded from, rather than accumulated on,\nthe XY chromosomes during male meiosis. Specifically, we revised the content as follows:\n\"Additionally, repressive histone modifications, H3K27 methylation and H2AK119ub, are also excluded from\nthe XY body during this period\" (page 3, lines 31–33).\n2. Line 62: The method used to prepare slides that preserve 3D nuclear structure after FACS cell isolation is\nnot described. Please include this information in the Methods section.\nResponses: We appreciate the reviewer for pointing this out. We have now added a detailed description in\nMethods, as follows:\n\"After FACS, sorted spermatogenic cells were collected by centrifugation at 300×g and washed once with\nPBS. The cell pellet was gently resuspended in fixation buffer (1% PFA with 0.15% Triton X-100).\nApproximately 1×10⁵ cells were resuspended in 10 µL fixation buffer and incubated at room temperature for\n5 min before being applied onto glass slides. The cell suspension was spread horizontally across the slide\n1\nusing the side of a 10 µL pipette tip to evenly distribute the cells. Slides were then air-dried and processed for\ndownstream staining.\" (page 41, lines 109–116).\n3. The authors appear to have used three different methods for microscopy analysis of male meiosis (FACS-\nisolated cells, tissue sections, and chromosome spreads), but it is not clear which method was used for each\nimage. Please clarify this in the figure legends.\nResponses: We thank the reviewer for the suggestion. In the revised manuscript, we have now specified the\nmicroscopy method used for each image (FACS-isolated cells, tissue sections, or chromosome spreads) in the\ncorresponding figure legends throughout the manuscript.\n4. The method for combining FISH and immunostaining is not described. Please include this information in\nthe Methods section.\nResponses: We have now added a detailed description as follows:\n\"Frozen testicular sections were used for RNA FISH analysis. RNA-FISH assays were performed according\nto the manufacturer's protocol provided by the FISH Kit for frozen sections (GenePharma). Briefly, after\nfixation in 4% paraformaldehyde, tissues were cryoprotected through a sucrose gradient, embedded in OCT,\nand sectioned at a thickness of 5 µm. Frozen sections were rehydrated by adding buffer B to each section and\nincubating at room temperature for 15 min. Sections were incubated with protease K at 37℃ for 20 min.\nSections were blocked by adding a blocking buffer at 37°C for 30 min and then denatured at 78°C for 8 min.\nAfter alcohol gradient dehydration, hybridization was carried out with a FISH probe in a humidified chamber\nat 37°C in the dark overnight. RiboBio designed and synthesized the 5.8S and 28S FISH probes. Sections\nwere washed three times with Wash Buffer I (4× SSC with 0.1% Tween-20, Sigma) and then with Wash\nBuffer II (2× SSC, Sigma) and Wash Buffer III (1× SSC, Sigma) at 42°C in the dark for 10 min. Following\nRNA-FISH, γH2AX immunostaining was carried out on the same slides. Sections were incubated with\nγH2AX primary antibody (Table S1) for 2 h at room temperature, washed three times with PBST, and then\nincubated with secondary antibody (Table S1) for 1 h. After three additional washes, slides were mounted\nwith VECTASHIELD (H-1000, Vector Laboratories). Images were acquired using an Andor Dragonfly\nspinning-disk confocal microscope.\" (page 42, lines 131–149).\n5. The live cell imaging method is not described. Specifically, how was CTD visualized in Fig. 6E? Was TBP\ndetected using GFP? Please clarify.\nResponses: CTD in Fig. 6E was visualized using GFP-tagged CTD, and purified TBP proteins were labeled\nwith 594 NHS-Ester. The condensate-formation assay was carried out in buffer containing 20 mM Tris (pH\n7.4), 150 mM NaCl, 10% glycerol, and 5 mM DTT, with phase-separation components mixed at the\nconcentrations indicated in the manuscript. Live-cell imaging was performed on an Andor Dragonfly spinning-\ndisk confocal microscope with a 40×oil-immersion objective. Excitation lasers used were: 405 nm for BFP-\nNPM1, 488 nm for GFP-CTD, 561 nm for 594 NHS-Ester–labeled TBP, and 637 nm for Cy5-labeled rRNA.\nThese methods have now been included in the Methods section (page 48, lines 318–341).\n6. Line 120: This description does not match the negative Pearson correlation values shown in Fig. 1F. Could\nyou revisit this?\nResponses: We apologize for the mistake. We have revised the sentence as follows:\nOriginal statement:\n\"Importantly, similar to the observations in mouse spermatocytes, we found that both FBL and NPM1\nmigrated to the XY body during human spermatogenesis (Fig.1F-G). NPM1 also covered the XY body in\nstage IV–X PACs (Fig. 1G).\"\nRevised statement:\n2\n\"Importantly, similar to the observations in mouse spermatocytes, we found that FBL localized specifically to\none corner of the XY body (Fig. 1F), whereas NPM1 migrated to and progressively covered the XY body in\nstage IV–X PACs during human spermatogenesis (Fig. 1G).\" (page 6, lines 120–123).\n7. Line 154: I do not observe any cytoplasmic signals for SENP3. Please review or clarify.\nResponses: We thank the reviewer for pointing this out. After re-examining the images, we agree that the\ncytoplasmic signals of SENP3 were not clearly detectable. We have revised the description accordingly:\nOriginal statement:\n\"…where almost no SENP3 and rRNA signals were detected in the nucleus from spermatogonia to PACs (Fig.\n2D-E). Instead, both SENP3 and rRNAs were largely located in the cytoplasm, with neither component\nmigrating to the XY body (Fig. 2D-E), indicating that the nuclear localization of SENP3 and rRNA depends\non NPM1.\"\nRevised statement:\n\"…where almost no SENP3 and rRNA signals were detected in the nucleus from spermatogonia to PACs (Fig.\n2D-E), indicating that the nuclear localization of SENP3 and rRNA depends on NPM1.\" (page 7, lines 153–\n155).\n8. Line 207: The donut-like signals are intriguing but could be a result of antibody penetration. Please provide\nmore details on the slide preparation methods for this data.\nResponses: We thank the reviewer for this insightful comment. The donut-like γH2AX signals were obtained\nfrom immunofluorescence analysis of paraffin-embedded testicular sections. Testes were fixed in 4% PFA\novernight, dehydrated, embedded in paraffin, and sectioned at 5μm. After deparaffinization, rehydration, and\nantigen retrieval, sections were permeabilized with 0.1% Triton X-100 and incubated with primary antibodies\novernight at 4°C, followed by Alexa Fluor-conjugated secondary antibodies. Nuclei were counterstained with\nDAPI, and images were acquired using an Andor Dragonfly spinning-disk confocal microscope and processed\nwith Imaris (v8.1).\nImportantly, we have repeated this experiment three biological replicates, and the donut-like γH2AX pattern\nwas consistently and specifically observed in Senp3SKO and Npm1SKO spermatocytes, but never in control\nsamples processed in parallel under identical staining conditions, suggesting that this phenotype is unlikely an\nartifact caused by incomplete antibody penetration.\nThese experimental details and clarifications have now been added to the Methods section of the revised\nmanuscript (page 41, lines 96–105).\n9. I am unclear on how the right panel of Fig. 4F was quantified. Could you please explain the quantification\nmethod used in the figure legend?\nResponses: We thank the reviewer for the comment. The quantification in the right panel of Fig. 4F was\nperformed as follows:\n1) A straight line was drawn across the region of interest using the ImageJ \"Plot Profile\" tool.\n2) ImageJ generated pixel-intensity plots for MDC1 (green) and rRNA (red) along the same trajectory.\n3) The resulting intensity values were exported and plotted in GraphPad Prism.\nThe following text has been added to the figure legend: \"Line-scan fluorescence quantification was performed\nusing ImageJ 'Plot Profile' along the indicated regions, and intensity values were plotted in GraphPad Prism.\"\n(page 31).\n3\n10. Line 507: Please specify the \"diplotene stage\" for clarity.\nResponses: We thank the reviewer for the suggestion. We have now specified the \"diplotene stage\" in the\nrevised main text for clarity (page 19, line 524).\n11. The model in Fig. 7I does not reflect DDR-dependent initiation of MSCI. I suggest revising it to more\nclearly illustrate the two steps: one involving DDR and the other nucleolar migration.\nResponses: We thank the reviewer for the valuable comment. We have revised Fig. 7I to further illustrate the\ntwo-step mechanism. First, DDR-dependent MSCI initiation, followed by nucleolar migration (Fig. R1A).\nThe updated model has been incorporated into the revised manuscript (Fig. 7I).\nFigure R1. Schematic models. A. Schematic model showing that SENP3, NPM1, and rRNAs migrate to the XY body\nand regulate MSCI through phase separation following DDR factors that initiate MSCI.\n12. For Fig. 6C, please include the number of independent experiments in the figure legend.\nResponse: We thank the reviewer for the suggestion. The figure legend for Fig. 6C now specifies the number\nof independent experiments: \"FRAP quantification of NPM1 in early (n=27) and mid-to-late (n=17) PACs\n(three biological replicates).\"(page 34).\nReviewer #2:\nRemarks to the Author:\nThe revised article by Li, et al., is improved relative to the original submission through addition of new data,\nrevision to tone down data interpretations and the addition of quantitation of the many fluorescence\nmicroscopy images. While improved, several issues need to be addressed before the manuscript could be\nconsidered for publication. The reviewer provides evaluations of the authors' point-by-point responses to their\noriginal comments, noting experiments that require further attention. With appropriate revision, the\nmanuscript will be of broad interest to the biomolecular condensates community as well as scientists in the\ndevelopmental biology field.\nResponses: We sincerely thank the reviewer for recognizing the substantial improvements in the revised\nmanuscript and for considering the study to be of broad interest to both the biomolecular condensates and\n4\ndevelopmental biology communities. We appreciate the reviewer's careful evaluation of our point-by-point\nresponses and address each specific concern in detail below.\nPage 29 of rebuttal, general responses to Reviewer 2. The revisions to figures to include quantitation and\nstatistics are acceptable.\nComment 1. See Comment 4, below.\nPage 5. \"These results demonstrate that the LLPS ability of NPM1 is required for excluding Pol II from the\nXY body in PACs.\" Suggest changing \"demonstrate\" to \"support\" to reflect the correlative nature of the data.\nResponses: We thank the reviewer for the suggestion. We have revised the sentence accordingly in the revised\nmanuscript (page 16, line 457).\nComment 2. The revision is acceptable.\nComment 3. The revisions are acceptable.\nComment 4. \"Additionally, live cell images showed that EGFP-NPM1 exhibited fusion behavior in the XY\nbody. These data indicate that NPM1 exhibits liquid-like features and undergoes LLPS in the XY body.\"\nThe still images in Fig. R1 B are suggestive of fusion by the NPM1 condensates but are not definitive. Perhaps\na video would be more convincing. The key events to capture are the rounding of an irregularly shaped\ncondensate after fusion. The rounding-up is evidence of surface tension, a property of fluids. If the authors\nhave access, they could possibly use lattice light sheet microscopy to capture fusion events with higher\nresolution with the added benefits of reduced light load (e.g., bleaching).\nC show the uniform recovery of fluorescence in a round condensate, the data in panel D appear grainy,\nsuggesting that the material properties of the XY body have changed. The authors need to address this\napparent difference. Also, scale bars are missing from these two panels.\nResponses: We thank the reviewer for the constructive comment. Following the recommendation, we have\nnow included a live-cell imaging video that directly captures fusion events of EGFP-NPM1 condensates in\nthe XY body of mid-to-late pachytene spermatocytes (Video R1). Fusion behavior is clearly observed.\nHowever, before and after fusion, the NPM1 condensates did not exhibit a round condensate morphology (Fig.\nR2A). Notably, this is consistent with the grainy appearance and slower fluorescence recovery of EGFP-NPM1\nsignals after photobleaching in mid-to-late pachytene spermatocytes (Fig. R2B, right), suggesting that the\nproperties of the XY body progressively change during pachytene progression. In contrast, the condensates in\nearly pachytene spermatocytes (Fig. R2B, left) exhibit uniform and rapid fluorescence recovery, indicative of\na more dynamic, liquid-like state. Taken together, these observations support a model in which an initially\nDDR-triggered liquid phase gradually gains increasingly stabilized or multivalent interactions as additional\nproteins accumulate, thereby transitioning toward a more gel-like state over time (Alavattam et al., 2021). We\nhave now integrated these results and this discussion into the revised manuscript (Video 1; page 13, lines 343–\n344; page 18, lines 493–503). Scale bars have also been added to the updated figures (Fig. R2B in the letter\nFig. 6A-B in the main text).\ncondensates into larger condensates in the XY body of Npm1EGFP PAC. Scale bars, 1μm. B. FRAP analysis of NPM1\nmobility in early and mid-to-late PACs of Npm1EGFP mice. The white dotted box indicating the FRAP region. The white\ndot circle indicating the XY body. Scale bars: PACs, 5µm; XY body, 1µm. C. FRAP quantification of NPM1 in early\n(n=27) and mid-to-late (n=17) PACs (three biological replicates).\nIn addition, following the reviewer's suggestion, we attempted long-term high-resolution live imaging using\na Luxendo MuVi-SPIM-CS lattice light-sheet microscope (Fig. R3A). Although this approach effectively\nreduced the fluorescence bleaching, the numerical aperture of the available detection objective (NA < 1.0)\nwas insufficient to clearly resolve the XY body or EGFP-NPM1 subnuclear condensates in spermatocytes (Fig.\nR3B). As a result, potential fusion events could not be definitively visualized using this system.\nR\nREDACTED\nfolded C-terminal domain of NPM1 binds nucleic acids, including rRNA. Grummitt, et al., showed that the\nW286A, W288A mutations cause this domain to unfold, abrogating interactions with rRNA and eliminating\nthe localization of NPM1 in the nucleolus. Deletion of the C domain was additionally shown by Mitrea, et al.\n(eLife, 2016), to abrogate nucleolar localization of NPM1. These effects are due to loss of rRNA binding, not\ndue to loss of hydrophobic interactions associated with phase separation. The C domain unfolds with these\nmutations due to loss of hydrophobic interactions in the core of the domain, destabilizing the folded structure.\nThe authors need to revise their statements in this regard.\nGiven this, it is puzzling that both WT and WW mutant NPM1 bind rRNA in Fig. R2 B. What are these RNA\nspecies and why is it that the mutant binds as well as WT? Is the assay probing only non-specific interactions?\nMitrea, et al., also showed that the C domain is required for binding rRNA in vitro (Fig. 10B).\nResponses: We thank the reviewer for the insightful comments. We have carefully revisited the studies by\nGrummitt et al. and Mitrea et al., and we appreciate the opportunity to clarify our interpretation.\nAs reported by Grummitt et al., the W286A/W288A mutations disrupt the hydrophobic core of the C-terminal\ndomain, leading to its unfolding and mislocalization of NPM1 to the nucleoplasm (Grummitt et al., 2008).\nThis is consistent with our observations in both 293T cells (Fig. R4A) and pachytene spermatocytes (Fig.\nR4B). Mitrea et al. further demonstrated that deletion of the C-terminal domain (NPM1N238) abolishes the\nability of NPM1 to form rRNA-containing condensates and disrupts nucleolar localization (Mitrea et al., 2016).\nHowever, Fig. 10B in Mitrea et al. cited by the reviewer (Fig. R4C) assessed turbidity/light-scattering\nproperties, an assay commonly used to measure LLPS behavior, rather than directly quantifying rRNA-\nbinding affinity. Thus, to our knowledge, neither Grummitt et al. nor Mitrea et al. directly examined whether\nthe NPM1W286A,W288A mutant retains any residual ability to bind rRNA.\nTo directly address this question, we performed EMSA experiments using high-purity wheat germ rRNA\n(BioWorld) and purified WT, NPM1W286A,W288A, and NPM1N238 proteins. Our results show that both\nNPM1W286A,W288A mutant and WT proteins displayed substantially strong rRNA binding, with shifted bands\ndetectable at 4 μM and nearly complete incorporation of rRNA into protein-RNA complexes at concentrations\nof 8 μM above (Fig. R4D). In contrast, the C-terminally truncated NPM1N238 protein exhibits minimal rRNA\nbinding below 8 μM and weak binding only at higher protein concentrations (Fig. R4D).\nTaken together, the W286A/W288A mutations do not abolish rRNA-binding capacity. Instead, we propose\nthat the loss of phase separation in the mutant is more likely attributable to the unfolding of the C-terminal\ndomain and consequent disruption of the hydrophobic pocket critical for condensate formation.\nimaging analysis for mCherry-tagged NPM1WT and mCherry-tagged NPM1W286A,W288A in transfected 293T cells. Nuclei\nwere counterstained with Hoechst (blue). Scale bars, 5μm. B. Double immunostaining of MDC1 (green) and\nNPM1W286A,W288A (red) in Npm1CKI PACs. Scale bars, 5μm. C. Light scattering assays of titrations of NPM1 constructs\ninto 50 μg/mL rRNA. The dashed line at 0.1 AU indicates the threshold, above which visible turbidity and microscopic\ndroplets can be detected (Mitrea et al., 2016).D. EMSA of the binding of NPM1WT, NPM1W286A,W288A and NPM1N238 to\nrRNA, using SYBR™ Gold-stained agarose gels. The concentrations for NPM1 and NPM1W286A,W288A used for lanes 1–\n8 were 0, 1, 2, 4, 8, 12, 16, and 20 µM, respectively. Quantification of the binding affinity of NPM1WT, NPM1W286A,W288A\nand NPM1N238 to rRNA. The binding curves (left) and KD values (right) of NPM1WT, NPM1W286A,W288A and NPM1N238\nto rRNA. The data were fitted to a dose-response curve using the software GraphPad Prism 9 to calculate the binding\naffinity between protein and rRNA.\nPage 3. \"Yet, the purified NPM1W286A,W288A proteins showed either a soluble state or fiber-like irregular\naggregates in vitro (Fig. R2C).\"The objects in Fig. R3 C labeled \"aggregates\" do not appear to have fibrillar\nfeatures and should not be referred to as such.\nResponses: We thank the reviewer for pointing this out. After re-examining the image, we agree that the\nstructures observed do not display fibrillar morphology and therefore should not be described as fiber-like.\nWe have revised the manuscript accordingly by removing the term “fiber-like” and now refer to these\nstructures simply as irregular aggregates.\nOriginal statement: \"However, purified NPM1W286A,W288A proteins exhibited either solution states or fiber-like\nirregular aggregates in vitro\".\nRevised statement: \"However, purified NPM1W286A,W288A proteins exhibited either solution states or irregular\naggregates in vitro\"(page 15, line 408).\nseparation ability.\"\nSuggested revision: \"These data strongly support the notion that the regulation of MSCI by NPM1 requires\nbinding to and phase separation with rRNA.\"\nResponses: We appreciate the reviewer's suggestion. As described above, our data in Fig. R4D show that the\nrRNA-binding ability of NPM1W286A,W288A mutant remains largely unaffected. Therefore, we felt our original\nstatement more accurately reflects the underlying mechanism.\nThe data in Fig. R6 are acceptable.\nPage 32. \"After 24 h treatment, the inhibitor was removed, and spermatocytes were collected at various time\npoints for chromosomal spreads to monitor NPM1 recruitment. By 0.5 h inhibitor post-removal, NPM1 was\nfirst recruited to the XY chromosome axes, inferred from the distribution of NPM1 and SYCP3 signals. By 1\nh, it had spread to XY chromatin. By 6 h, the NPM1 signal recovered to approximately 60% of the level in\nDMSO treated controls (Fig. R29C-D). These results suggest that NPM1 recruitment to the XY chromosomes\nfollows and mirrors the dynamics of DDR factors, such as MDC1 (Fig. R29C), which initially localize to the\nXY chromosomal axes and subsequently spread to chromatin loops (Turner, 2015).\"\nFig. R29 C. The recovery of MDC1 and NPM1 in this sequence of images requires quantitation. Also, the\nauthors might also quantify the recovery of rRNA.\nResponses: We thank the reviewer for the helpful suggestion. We have now performed additional\nimmunofluorescence analyses and quantitative measurements.\nUsing a monoclonal antibody against the 5.8S rRNA sequence (Novus, NB100-662SS), which detects all pre-\nrRNAs containing the 5.8S rRNA sequence, we found that rRNA began to reappear on the XY body at 1 h\nafter inhibitor removal. At this early stage, the signal was initially enriched in the dense-body region of the\nXY body and subsequently spread across the entire XY chromatin (Fig. R5A). This pattern differs from that\nof NPM1, which was first detected on the XY chromosome axes by 0.5 h post-removal (Fig. R5B). We further\nquantified the fluorescence recovery of MDC1, NPM1, and rRNA at each time point relative to their respective\nDMSO-treated controls. By 6 h after inhibitor removal, NPM1 and rRNA had both recovered to approximately\n60% of control levels, whereas MDC1 recovered to approximately 75% (Fig. R5C).\nTaken together, these analyses indicate that MDC1 and NPM1 are recruited to the XY body earlier during\nrecovery, while rRNA returns slightly later. We have included these results in the revised paper (Fig. 5F;\nExtended Data Fig. 11E and pages 12-13, lines 322-328).\nin a medium containing DMSO for 24 h, incubated in a medium containing ATRi for 24 h, and then incubated in a\nmedium without ATRi for the indicated time. Representative images of mid-PAC chromosomal spreads immunostained\nwith antibodies raised against SYCP3, MDC1 and rRNA(A) or NPM1(B). Nuclei were counterstained with DAPI (blue).\nHighlight the XY body with a white dotted box. Representative images and line quantifications showing the distribution\nof SYCP3 (grey), rRNA or NPM1 (red), and MDC1 (green). C. Quantification of NPM1, rRNA and MDC1 fluorescence\nintensity recovery in the ATRi treatment group, relative to the DMSO group.\nComment 5. Fig. R30 A. The authors should also show imaging of rRNA as the condensates change over time.\n\"In addition, the condensates underwent fusion on contact and recovered rapidly after photobleaching (10 s\nto recover 80%) (Fig. R30A, C).\"\nFusion events are not evident in these images. The authors need to present images that clearly indicate\ncondensate fusion events.\nstrengthened by presentation of quantitative analyses of the changes that occur in these condensates. Also,\nthe authors should illustrate the starting point of the experiment and the entire time sequence.\nResponses: We thank the reviewer for these valuable suggestions. In response, we performed additional\nimaging experiments using Cy5-labeled 5.8S rRNA produced by in vitro transcription and carried out a\ncomplete time-course analysis of the five-component condensate system.\nTo precisely define the starting point, we first generated condensates of TBP and Pol II CTD with RNA, as\nwell as NPM1 and rRNA. The time 0 was defined as the moment when NPM1-rRNA condensates were added\nto TBP–CTD condensates, followed by gentle tapping and immediate transfer to glass-bottom dishes. Despite\nminimizing handling steps, this process required ~30 seconds; thus the earliest frames correspond to the state\nimmediately after mixing.\nThese new experiments revealed that the five components rapidly reorganized into a distinct condensate\nmorphology, in which TBP and Pol II CTD localized to the condensate periphery, while NPM1 and rRNA\nsegregated to the interior (Fig. R6A). Importantly, Cy5-rRNA imaging allowed us to track rRNA distribution\nthroughout the entire time course. NPM1 and rRNA remained stably colocalized and exhibited no noticeable\nstructural changes during the experiment. To quantify the expulsion of Pol II CTD, we measured CTD\nfluorescence intensity normalized to TBP intensity within regions where CTD initially colocalized with TBP\ncondensates. Normalization to TBP controlled for variability in condensate size and imaging conditions. Using\nthis metric, CTD signal progressively decreased to ~15% of its initial value over 18 min (Fig. R6A-B; Video\nR2). In contrast, NPM1 and rRNA signals remained constant within the condensates, supporting the selective\nincompatibility between TBP/CTD and NPM1/rRNA.\nWe also captured clear fusion events. NPM1-rRNA condensates underwent fusion upon contact both before\nand after CTD release (Video R3 and Video R4), and FRAP measurements showed rapid recovery (80% within\n30 seconds) (Fig. R6C), indicating a liquid-like nature. Interestingly, NPM1-rRNA condensates were able to\npenetrate TBP condensates and fuse, but the resulting merged structures often displayed irregular shapes rather\nthan forming perfect spheres (Video R4). This may help explain why NPM1-EGFP condensates in the XY\nbody in vivo are also not strictly spherical. Notably, the maximal concentration of in vitro transcribed Cy5-\nlabeled 5.8S rRNA was 40μg/ml. Under this lower rRNA concentration, NPM1-rRNA condensates became\nvisibly smaller and the expulsion of Pol II CTD proceeded more slowly than that under higher rRNA\nconcentration using unlabeled rRNA. This concentration dependence is consistent with a requirement for\nsufficient rRNA to efficiently drive remodeling of TBP/CTD condensates. All new imaging data,\nquantifications, and representative time-course sequences have been incorporated into the revised manuscript\n(Extended Data Fig. 12A-C and pages 13-14, lines 356-365).\nliquid-like features in vitro. A. In vitro condensate formation live images of condensates formed by TBP (red),\nRNA Pol II CTD (green), rRNA (purple) and NPM1 (blue). Scale bar, 1μm. B. Quantification of rRNA/NPM1 and\nCTD/TBP relative fluorescence intensity. C.FRAP images of condensates formed by TBP, RNA Pol II CTD, rRNA\nand NPM1, and quantification (right). Scale bar, 1μm.\nComments 6 & 7. The revision is acceptable.\nComment 8. The new data with RNA Pol I inhibitors strengthens the manuscript.\nReferences:\n1. Alavattam, K. G., Maezawa, S., Andreassen, P. R., & Namekawa, S. H. (2021). Meiotic sex chromosome inactivation\nand the XY body: a phase separation hypothesis. Cell Mol Life Sci, 79(1), 18. doi:10.1007/s00018-021-04075-3\n2. Grummitt, C. G., Townsley, F. M., Johnson, C. M., Warren, A. J., & Bycroft, M. (2008). Structural consequences of\nnucleophosmin mutations in acute myeloid leukemia. J Biol Chem, 283(34), 23326-23332.\ndoi:10.1074/jbc.M801706200\n12\n3. Mitrea, D. M., Cika, J. A., Guy, C. S., Ban, D., Banerjee, P. R., Stanley, C. B., . . . Kriwacki, R. W. (2016).\nNucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear\nmotifs and rRNA. Elife, 5. doi:10.7554/eLife.13571\n13"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have gone a long way to address this reviewer's concerns regarding the R1 version of the manuscript and the\nmanuscript is now suitable for publication. The authors should be commended for their patience and hard work during the\nreview process, which has strengthened their scientific story and improved the manuscript.\nOne minor comment:\n1. Fig. R4, C need a legend for the NPM1 constructs. There's plenty of white-space in the graph for this."}, "author_response": ""}]

Decision letter