Transcriptional pausing induced by ionizing radiation enables the acquisition of radioresistance in nasopharyngeal carcinoma.

Lesions on the DNA template can impact transcription via distinct regulatory pathways. Ionizing radiation (IR) as the mainstay modality for many malignancies elicits most of the cytotoxicity by inducing a variety of DNA damages in the genome. How the IR treatment alters the transcription cycle and whether it contributes to the development of radioresistance remain poorly understood. Here, we report an increase in the paused RNA polymerase II (RNAPII), as indicated by the phosphorylation at serine 5 residue of its C-terminal domain, in recurrent nasopharyngeal carcinoma (NPC) patient samples after IR treatment and cultured NPC cells developing IR resistance. Reducing the pool of paused RNAPII by either inhibiting TFIIH-associated CDK7 or stimulating the positive transcription elongation factor b, a CDK9-CycT1 heterodimer, attenuates IR resistance of NPC cells. Interestingly, the poly(ADP-ribosyl)ation of CycT1, which disrupts its phase separation, is elevated in the IR-resistant cells. Mutation of the major poly(ADP-ribosyl)ation sites of CycT1 decreases RNAPII pausing and restores IR sensitivity. Genome-wide chromatin immunoprecipitation followed by sequencing analyses reveal that several genes involved in radiation response and cell cycle control are subject to the regulation imposed by the paused RNAPII. Particularly, we identify the NIMA-related kinase NEK7 under such regulation as a new radioresistance factor, whose downregulation results in the increased chromosome instability, enabling the development of IR resistance. Overall, our results highlight a novel link between the alteration in the transcription cycle and the acquisition of IR resistance, opening up new opportunities to increase the efficacy of radiotherapy and thwart radioresistance in NPC.

A chaperone-like function of FUS ensures TAZ condensate dynamics and transcriptional activation.

The Hippo pathway has important roles in organ development, tissue homeostasis and tumour growth. Its downstream effector TAZ is a transcriptional coactivator that promotes target gene expression through the formation of biomolecular condensates. However, the mechanisms that regulate the biophysical properties of TAZ condensates to enable Hippo signalling are not well understood. Here using chemical crosslinking combined with an unbiased proteomics approach, we show that FUS associates with TAZ condensates and exerts a chaperone-like effect to maintain their proper liquidity and robust transcriptional activity. Mechanistically, the low complexity sequence domain of FUS targets the coiled-coil domain of TAZ in a phosphorylation-regulated manner, which ensures the liquidity and dynamicity of TAZ condensates. In cells lacking FUS, TAZ condensates transition into gel-like or solid-like assembles with immobilized TAZ, which leads to reduced expression of target genes and inhibition of pro-tumorigenic activity. Thus, our findings identify a chaperone-like function of FUS in Hippo regulation and demonstrate that appropriate biophysical properties of transcriptional condensates are essential for gene activation.

Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage.

DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9-cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid-liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb-PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.

Silkworm serpin32 functions as a negative-regulator in prophenoloxidase activation.

The extracellular serine protease cascade is an essential component of insect humoral immunity. Serine protease inhibitors (serpins) play an important regulatory role in the process of insect immunity by regulating the serine protease cascade pathway. We aimed to clarify the function of Bmserpin32 in this study. First, we performed homologous sequence alignment and phylogenetic analysis of Bmserpin32. Bmserpin32 was found to share 64% amino acid sequence identity with Manduca sexta serpin7, an immunomodulatory protein. Bmserpin32 cDNA was cloned, and the recombinant Bmserpin32 protein was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity and gel filtration chromatography. The activity assay showed that Bmserpin32 had significant inhibitory activity against trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and site-directed mutagenesis combined with activity assays indicated that the cleavage site of Bmserpin32 is between Arg359 and Ile360. After infection with E. coli or Micrococcus luteus, the expression level of Bmserpin32 in immune-related tissues was significantly upregulated. In addition, Bmserpin32 could delay or inhibit the melanization of hemolymph by inhibiting the activation of prophenoloxidase in larval hemolymph. Furthermore, a physiological target of Bmserpin32 was identified as the clip protease, BmPAP3, an apparent ortholog of M. sexta propenoloxidase-activating protease-3. Our observations enable a better understanding of the physiological role of Bmserpin32 in regulating melanization in silkworm.