High-precision mapping of nuclear pore-chromatin interactions reveals new principles of genome organization at the nuclear envelope
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eLife assessment
This manuscript describes important findings that will impact our understanding of nuclear pore complex (NPC)-genome interactions and how nuclear pore proteins (nucleoporins) impact super enhancer function. The authors develop a clever new approach termed NPC-DamID to map chromatin-NPC interactions that has several important advantages over existing techniques and may be used in contexts that are incompatible with genetic manipulation. The authors provide compelling evidence that nucleoporins interact with super enhancers in multiple cell types and also suggest that the phase separation behavior of nucleoporins contributes to the hierarchical organization of super enhancers, which is exciting and will stimulate additional work.
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Abstract
The role of nuclear pore complexes (NPCs) in genome organization remains poorly characterized due to technical limitations in probing genome-wide protein-DNA interactions specific to the nuclear periphery. Here, we developed a new sensitive method, NPC-DamID, which combines in vitro reconstitution of nuclear import and DamID technology. The fixation-free method identifies chromatin interactions at the NPCs in intact nuclei from cells and tissues. We found that NPCs are preferentially associated with common and hierarchically arranged super-enhancers (SEs) across multiple cell types. We also uncovered phase-separated condensates at NPCs that compartmentalize and concentrate transcriptional coactivators and structural proteins at SE-regulated genes. Our results support NPCs as anchoring sites for SE regulatory hubs and cell-type-specific transcriptional control.
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eLife assessment
This manuscript describes important findings that will impact our understanding of nuclear pore complex (NPC)-genome interactions and how nuclear pore proteins (nucleoporins) impact super enhancer function. The authors develop a clever new approach termed NPC-DamID to map chromatin-NPC interactions that has several important advantages over existing techniques and may be used in contexts that are incompatible with genetic manipulation. The authors provide compelling evidence that nucleoporins interact with super enhancers in multiple cell types and also suggest that the phase separation behavior of nucleoporins contributes to the hierarchical organization of super enhancers, which is exciting and will stimulate additional work.
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Joint Public Review:
As nucleoporins can function at intact nuclear pore complexes (NPCs) or outside of NPCs as individual proteins or subcomplexes, it remains challenging to molecularly define the pool of molecules that exert a specific function. To address this challenge, here the authors develop a new method for specifically mapping NPC-associated loci by DamID with a recombinant fusion protein of the Dam methylase and the nuclear transport receptor, importin b (Dam-Impb), in permeabilized cells. The authors demonstrate that Dam-Impb is active, accumulates at the NPC and, using super-resolution microscopy, methylates NPC-adjacent regions; other observations further support the assertion that the approach is specific for NPC-associate chromatin regions. Furthermore, NPC-DamID does not require genetic manipulation and they show that it …
Joint Public Review:
As nucleoporins can function at intact nuclear pore complexes (NPCs) or outside of NPCs as individual proteins or subcomplexes, it remains challenging to molecularly define the pool of molecules that exert a specific function. To address this challenge, here the authors develop a new method for specifically mapping NPC-associated loci by DamID with a recombinant fusion protein of the Dam methylase and the nuclear transport receptor, importin b (Dam-Impb), in permeabilized cells. The authors demonstrate that Dam-Impb is active, accumulates at the NPC and, using super-resolution microscopy, methylates NPC-adjacent regions; other observations further support the assertion that the approach is specific for NPC-associate chromatin regions. Furthermore, NPC-DamID does not require genetic manipulation and they show that it can be applied to both diverse cell lines as well as tissues. The authors confirm the association of nucleoporins with super-enhancers (SEs) in line with their prior work, now confirmed to occur at NPCs based on this study. Among SEs categorized as hierarchical enhancers (Nat Commun 9, 943 (2018)), hub enhancers are over-represented for methylation by Dam-Impb. The association of such enhancers with cohesin and CTCF suggests these regions could have a critical role in chromatin folding; enhancer-associated factors and marks such as H3K27 acetylation, RNA polymerase II, P300, CTCF and BRD4 also enrich at Dam-Impb methylation peaks. Using proximity ligation, the authors provide further evidence that Tpr, which interacts with the NPC basket, colocalizes with CTCF, BRD4 and P300. Based on these observations, the authors hypothesized that nucleoporin phase separation at SEs might potentiate phase separation of other factors at these elements. Consistent with previous work, over-expression of the intrinsically disordered region (IDR) of Nup153, a component of the NPC basket, forms nuclear droplets that are largely dispersed by 1,6-hexanediol. In this same condition, colocalization RNAPII and both Tpr and BRD4 is reduced, although some interactions between IDRs were not sensitive to this treatment. Last, using a lac operator array as a tethering site, the authors show that tethered Nup153 IDR recruits the carboxy terminal domain of RNAPII and Med1. However, whether the biology of how nucleoporins at NPCs influence SEs depends on biomolecular condensation will require future study.
Overall, the reviewers agree that this is an excellent manuscript that will impact our understanding of nuclear pore complex-genome interactions and how nucleoporins impact super enhancer function. The data are generally of high quality and are reasonably interpreted. There are, however, several important controls or analyses that would strengthen the conclusions of the paper, as outlined below.
1. NPC vs nucleoplasmic interactions: One of the main claims of the paper is that it provides a way to study specifically the NPC-associated loci and contrast them to the nucleoplasmic Nup-associated loci. Unfortunately, the authors do not devote much space to this comparison and many of the manipulations involve proteins that are in both locations (see below). This seems like an important, missed opportunity. The choices of Tpr or Nup153 should be more clearly justified. The Dpn8 staining appeared in regions outside of the nuclear envelope, which is inconsistent with the text. This should be addressed.
2. The PLA experiments: Although Tpr exists both at the NPC and in the nucleoplasm, the authors interpret these experiments as if they are exclusively reporting on proximity of enhancer proteins to the NPC. The images (e.g. Figure 5a, supplementary Figure 5) make it clear that the foci are throughout the nucleus. Where are the antigens recognized by the antibodies to Tpr and what this may mean for the findings? Further, PLA experiments are prone to artefacts and, while the authors have included a knockdown of Tpr as a negative control, additional controls would strengthen their conclusions. For example, what is the result when Tpr colocalization with NPC-specific proteins is assessed? How is that affected by hexanediol? A better PLA experiment might be to assess colocalization of Dam-ImpB or Dpn8 (bound to Dam-ImpB methylated sites) with super enhancer proteins such as Med1, CTCF, Brd4, etc. With regards to the PLA with
3. Analysis of genomic data: The normalization of the DNA sequencing tracks is not sufficiently explained. Moreover, some of the correlations using meta-site plots are not convincing. For example, the peaks of Nup153 or Nup98 methylation over Imp-B peaks are apparently weak. Although the authors report local maxima, these may not be strong associations. This raises the possibility that the stronger Nup153 or Nup98 peaks are not ImpB peaks. A better way to test for this would be to correlate the ImpB peak intensity to the Nup153 or Nup98 peak intensity globally. The expectation is that there will be both correlated peaks that show strong methylation by Nup153/Nup98 and ImpB, as well as peaks that do not (i.e. those in the nucleoplasm). Along these lines, the Dam alone control can be used for comparison. Peaks identified by Dam alone should not be correlated with ImpB, Nup153, Nup98, CTCF, RNAPII, Cohesin, H3K27Ac, Brd4, Mediator, super enhancers, hubs, etc. Also, what is the source of the Nup93 CUT&RUN data? It was unclear if it was from this study or a prior publication.
4. FISH experiments: these should be in the main figures of the paper and better described. How many loci were assessed in each category? Are the differences between the three classes significant? Also, the order of the legend is the opposite of the order of the bar segments, which is confusing to the reader. Related to Figure 2j: What are the FISH probes used here? How many cells were quantified?
5. The focus on IDRs as the primary functional mechanism for the NPC-SE connection was felt to be the least well-justified of the authors' conclusions. In particular, the quantitative effects in Fig. 6 are over-stated while caveats including possible over-expression artifacts and changes in the nuclear concentration of the IDRs due to efflux out of the nucleus in response to 1,6 hexanediol treatment as a consequence of the effect on the barrier of NPCs are not addressed. Additional experimental follow-up - for example does critical depletion of Nup153 (now possible with auxin degrons) disrupt the NPC-DamID profile? - would strengthen the support for the model.
6. Recent evidence points to the fact that 1,6-HD treatment probes the presence of hydrophobic interactions, rather than distinguishing between LLPS and interactions with spatially clustered binding sites (ICBS). These possibilities should be taken into account when interpreting the data, and should be discussed more thoroughly.
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