Dataset Information

HSPA1A and DNAJB1 regulate NELF condensate reversibility to safeguard transcriptional recovery under heat stress

ABSTRACT: The negative elongation factor (NELF) is essential in establishing RNA Polymerase II (Pol II) pausing at promoter-proximal regions, providing a key regulatory checkpoint for precise gene expression control. In response to heat shock (HS) stress, NELF has been observed to form nuclear condensates, but the mechanisms that govern their dynamic behavior and functional significance during stress adaptation remain poorly understood. Here, we leverage a nanobody-based proximity labeling (NbPro) strategy to characterize the composition and regulation of HS-induced NELF condensates. Through this approach, we identify molecular chaperones HSPA1A and DNAJB1 as key regulators that maintain the dynamic reversibility of NELF condensates. While dispensable for HS-induced transcriptional downregulation, this process is essential for transcriptional restart during recovery. Mechanistically, DNAJB1 recognizes NELFA and facilitates the recruitment of HSPA1A into NELF condensates. This coordinated action prevents NELF condensates from transitioning into aggregates and ensures their reversible disassembly during recovery. We further show that NELFA undergoes a tightly regulated phosphorylation-dephosphorylation cycle during HS. Disruption of NELF condensate dynamics leads to sustained NELFA phosphorylation, reduced chromatin binding, impaired Pol II pausing, and the release of non-productive Pol II complexes, ultimately compromising transcriptional recovery. Collectively, our findings reveal a critical chaperone-dependent mechanism that governs NELF condensate dynamics and highlight promoter-proximal pausing not merely as a means of transcriptional repression, but as a checkpoint to prevent immature Pol II escape and ensure productive transcriptional restart following heat shock.

ORGANISM(S): Homo Sapiens

SUBMITTER: Huasong Lu

PROVIDER: PXD064175 | iProX | Wed May 21 00:00:00 BST 2025

REPOSITORIES: iProX

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HSPA1A and DNAJB1 regulate NELF condensate dynamics to safeguard transcriptional recovery under heat stress.

Jiang Shuyao S Jia Zixuan Z Zhu Wenxuan W Liu Yu Y Fu Huanyi H Zhu Feifeng F Li Zhuo Z Yang Jiaye J Zhu Yiying Y Sun Zhongxing Z Zhu Tianyi T Quan Xuebo X Jiao Huipeng H Huang Kai K Wu Zhibing Z Zou Wei W Yang Bing B Lu Yi Y Zhang Long L Zhou Fangfang F Fang Dong D Lu Huasong H

Molecular cell 20260206

Promoter-proximal pausing by negative elongation factor (NELF) establishes a critical checkpoint for RNA polymerase II (RNA Pol II) transcription. Heat shock (HS) induces NELF to form nuclear condensates, yet how their dynamics are regulated and coupled to transcriptional adaptation remains unclear. Using a nanobody-based proximity labeling strategy (NbPro), we identify the molecular chaperones HSPA1A and DNAJB1 as key regulators of NELF condensate dynamics. Although dispensable for initial HS-i ...[more]

PMID: 41653920

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Project description:Like tobacco smoking, habitual marijuana smoking causes numerous adverse pulmonary effects. However, the mechanisms of action involved, especially as compared to tobacco smoke, are still unclear. To uncover putative modes of action, this study employed a toxicogenomics approach to compare the toxicological pathways perturbed following exposure to marijuana and tobacco smoke condensate in vitro. Condensates of mainstream smoke from hand-rolled tobacco and marijuana cigarettes were similarly prepared using identical smoking conditions. Murine lung epithelial cells were exposed to low, medium and high concentrations of the smoke condensates for 6 hr. RNA was extracted immediately or after a 4-hr recovery period and hybridized to mouse whole genome microarrays. Tobacco smoke condensate (TSC) exposure was associated with changes in xenobiotic metabolism, oxidative stress, inflammation, and DNA damage response. These same pathways were also significantly affected following marijuana smoke condensate (MSC) exposure. Although the effects of the condensates were largely similar, dose-response analysis indicates that the MSC is substantially more potent than TSC. In addition, steroid biosynthesis, apoptosis, and inflammation pathways were more significantly affected following MSC exposure, whereas m-phase cell cycle pathways were more significantly affected following TSC exposure. MSC exposure also appeared to elicit more severe oxidative stress than TSC exposure, which may account for the greater cytotoxicity of MSC. This study shows that in general, MSC impacts many of the same molecular processes as TSC. However, subtle pathway differences can provide insight into the differential toxicities of the two complex mixtures. Murine epithelial lung cells were exposed to tobacco smoke condensates (0, 25, 50, 90 Î¼g/ml) or marijuana smoke condensates (0, 2.5, 5, 10 Î¼g/ml) in serum-free medium for a six hour period. Following the six-hour exposure, cells were either harvested immediately or washed with phosphate-buffered saline and incubated in fresh serum-free medium for a four hour recovery period. Total RNA was extracted from the cells and hybridized against Universal Mouse Reference RNA (Agilent Technologies Canada, Inc.) to Agilent whole mouse genome microarray slides containing 44,000 transcripts. A LOWESS normalization was applied to expression results, and statistically significant genes were identified using the R library MAANOVA. Microarray results were validated by real time RT-PCR.

Dataset Information

HSPA1A and DNAJB1 regulate NELF condensate reversibility to safeguard transcriptional recovery under heat stress

Publications

HSPA1A and DNAJB1 regulate NELF condensate dynamics to safeguard transcriptional recovery under heat stress.

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