Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Maintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin-preferentially ∼30 bp upstream of transcription start-sites-and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.

Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.

Liver X receptors (LXRs) have been proposed to have some anticancer properties, through molecular mechanisms that remain elusive. Here we report for the first time that LXR ligands induce caspase-1-dependent cell death of colon cancer cells. Caspase-1 activation requires Nod-like-receptor pyrin domain containing 3 (NLRP3) inflammasome and ATP-mediated P2 Â 7 receptor activation. Surprisingly, LXRb is mainly located in the cytoplasm and has a non-genomic role by interacting with pannexin 1 leading to ATP secretion. Finally, LXR ligands have an antitumoral effect in a mouse colon cancer model, dependent on the presence of LXRb, pannexin 1, NLRP3 and caspase-1 within the tumor cells. Our results demonstrate that LXRb, through pannexin 1 interaction, can specifically induce caspase-1-dependent colon cancer cell death by pyroptosis.

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

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Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF-β1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB-crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB-crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad-dependent pathway. We demonstrate here that αB-crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB-crystallin-deficient mice are protected from bleomycin-induced fibrosis. Similar protection from fibrosis was observed in αB-crystallin KO mice after transient adenoviral-mediated over-expression of IL-1β or TGF-β1. We show in vitro in primary epithelial cells and fibroblasts that αB-crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF-β1-Smad pathway and the consequent activation of TGF-β1 downstream genes. αB-crystallin over-expression disrupts Smad4 mono-ubiquitination by interacting with its E3-ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB-crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono-ubiquitination and nuclear export are favoured and thus TGF-β1-Smad4 pro-fibrotic activity is inhibited. This study demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases.