In eukaryotes, the transcription process by RNA polymerase II (RNA Pol II) is tightly regulated. Shortly after initiation, RNA Pol II often pauses near the promoter, a phenomenon known as “promoter-proximal pausing”. This pausing is jointly maintained by the negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF), playing a crucial role in precisely controlling gene expression. Previous studies have shown that heat stress induces the formation of nuclear condensates by NELF, but the dynamic regulatory mechanisms of these condensates, their connection to transcriptional adaptation, and the specific role of NELF in heat stress-induced transcriptional repression remain unclear and controversial.

Moreover, traditional proximity labeling techniques face limitations when studying biomolecular condensates under stress conditions, including interference from fusion tags on protein function, overly large labeling radii, and low temporal resolution, which hinder precise profiling of the condensate proximity proteome. Recently, the Lu Huasong team at Zhejiang University published in Molecular Cell, developing a novel proximity labeling technique called NbPro. They identified HSPA1A and DNAJB1 as core regulatory factors of NELF condensates. Through specific binding, these factors maintain the dynamic reversibility of NELF condensates, regulate NELFA phosphorylation cycles and chromatin association, stabilize RNA Pol II promoter-proximal pausing, and ensure efficient transcriptional recovery following heat stress.

Figure 1. Graphical abstract

Developing the Novel Proximity Labeling Technique NbPro for Precise Capture of Molecules within Condensates

To overcome the shortcomings of traditional APEX2 fusion labeling, the team developed a nanobody-based proximity labeling strategy called NbPro. By fusing an anti-ALFA nanobody with APEX2, the approach targets ALFA-tagged proteins of interest after cell fixation to achieve in situ biotinylation. Experiments confirmed that NbPro-1 exhibits stronger peroxidase activity, and adding sucrose and DTT effectively reduces the labeling radius. NbPro eliminates interference with protein function seen in conventional methods, improves spatial resolution, and serves as a reliable tool for dissecting the proximity proteome of biomolecular condensates.

Figure 2. Establishment of NbPro

Identification of Core Components of NELF Condensates under Heat Stress: HSPA1A and DNAJB1

Using NbPro, the proximity proteome of NELFA condensates was analyzed under heat stress (HS) and non-heat stress (NHS) conditions, identifying 728 specifically enriched proteins. GO enrichment analysis revealed strong enrichment in processes such as chromatin binding and transcriptional elongation. Proximity ligation assays (PLA) and fluorescence colocalization experiments further confirmed tight binding of the molecular chaperones HSPA1A and DNAJB1 to NELFA under heat stress, establishing them as core components of heat stress-induced NELF condensates.

Figure 3. NbPro analysis of NELF condensates under HS stress

The HSF1-HSPA1A/DNAJB1 Axis Maintains Liquid-like Dynamics of Condensates

Experiments revealed that although NELF and HSF1 condensates are spatially independent, knocking down HSF1 (KD) does not prevent heat stress-induced NELF condensate formation but causes persistent condensates during recovery that fail to dissolve. Treatment with 1,6-hexanediol and FRAP assays showed that disruption of the HSF1-HSPA1A/DNAJB1 axis renders NELF condensates resistant to hexanediol and markedly reduces NELFA mobility, indicating a transition from liquid to gel- or solid-like state. These findings demonstrate that HSF1 induces expression of the molecular chaperones HSPA1A and DNAJB1 to maintain the liquid-like dynamics and reversibility of NELF condensates, preventing pathological aggregation.

Figure 4. HSF1-HSPA1A/DNAJB1 axis maintains reversibility of HS-induced NELF condensates

DNAJB1 Mediates HSPA1A Recruitment and Phase Separation Inhibition

Using LacO/LacI tethering experiments, researchers found that the tentacle domain of NELFA is responsible for recruiting molecular chaperones. Further studies confirmed that the C-terminal domain (CTD) of DNAJB1 binds this tentacle domain, thereby mediating HSPA1A recruitment. In vitro droplet formation assays showed that HSPA1A and DNAJB1 synergistically suppress liquid-liquid phase separation of NELFA and enhance droplet fluidity. As a co-chaperone, DNAJB1 binds the NELFA tentacle domain and recruits HSPA1A to coordinately regulate the dynamic properties of NELF condensates.

Figure 5. Interaction between DNAJB1 and NELFA tentacle domain promotes HSPA1A integration into NELF condensates

Condensate Dynamics Regulate NELFA Phosphorylation Cycles

Phos-tag gel analysis revealed dynamic, MEK1/2 pathway-dependent phosphorylation of NELFA induced by heat stress. Experiments showed that loss of HSPA1A/DNAJB1 function, which impairs condensate dynamics (preventing dissociation), significantly blocks NELFA dephosphorylation during recovery. HSPA1A/DNAJB1 regulate NELF condensate dissociation to facilitate phosphatase access to NELFA, thereby maintaining balanced phosphorylation cycles.

Figure 6. Impaired NELF condensate dynamics lead to persistent NELFA phosphorylation

NELF Condensates Serve as Transcriptional Quality Control Checkpoints to Ensure Recovery

By constructing an NELFA 16A mutant (mutating 16 potential phosphorylation sites), combined ChIP-Rx, TT-seq, and GRO-seq analyses showed that DNAJB1 knockout or HSP70 inhibition weakens NELF promoter binding, causing aberrant escape of RNA Pol II from pause sites. These escaped complexes are non-productive, ultimately leading to significantly reduced transcriptional completion rates after heat stress. NELF-mediated pausing is not merely transcriptional repression but acts as a quality control checkpoint, preventing aberrant escape of immature RNA Pol II to safeguard transcriptional fidelity.

Figure 7. Chaperone-dependent NELF condensate dynamics are essential for NELF-chromatin binding and transcriptional recovery after HS

NELF Condensate Dynamics Regulate Transcriptional Recovery; Loss of HSPA1A/DNAJB1 Function Causes Aberrant RNA Pol II Escape and Impaired Recovery

5-ethynyluridine (5-EU) labeling and transient transcriptome sequencing (TT-seq) experiments showed that NELF is not required for initial transcriptional repression during heat stress but is critical for transcriptional reactivation during recovery. DNAJB1 deficiency disrupts the NELF-mediated “pausing” mechanism, allowing RNA Pol II to aberrantly escape into gene bodies. These escaped Pol II complexes are non-productive, preventing recovery of the elongation index (EI) and severely impairing transcriptional recovery. This indicates that NELF-mediated pausing serves as a key checkpoint for transcriptional fidelity, preventing aberrant escape of immature RNA Pol II to ensure productive transcriptional restart.

Figure 8. Disruption of NELF-mediated RNA Pol II pausing causes immature RNA Pol II to escape from pause sites, impairing productive transcriptional restart after HS

Conclusion

This study developed the novel proximity labeling technique NbPro, overcoming functional interference from traditional fusion tags and enabling high-resolution in situ profiling of biomolecular condensates under stress conditions. The work uncovers the HSF1-HSPA1A/DNAJB1-NELF regulatory axis, confirming that molecular chaperones maintain the liquid-like dynamics and reversibility of NELF condensates by binding the NELFA tentacle domain and balancing phosphorylation cycles.

abinScience Experimental Support

abinScience provided Benzonase nuclease for the study. It was used in chromatin fraction immunoprecipitation (Chromatin fraction IP) experiments to efficiently digest chromatin-associated nucleic acids, facilitating the isolation and enrichment of chromatin fragments bound to target proteins and providing essential technical support for subsequent immunoprecipitation and protein interaction analyses.