Improving CAR-T immunotherapy for lymphoma by fine-tuning avidity

Transcription elongation by RNA polymerase II (Pol II) is an integral step in eukaryotic gene expression. The speed of Pol II is controlled by a multitude of elongation factors, but the regulatory mechanisms remain incompletely understood, especially for higher eukaryotes. In this work, we developed a single-molecule platform to visualize the dynamics of in vitro reconstituted mammalian transcription elongation complexes (ECs). This platform enabled us to follow the elongation and pausing behavior of EC in real time and dissect the role of each elongation factor in the kinetic control of Pol II. We found that the mammalian EC harbors multiple gears depending on its associated factors and phosphorylation status. The elongation factors are not functionally redundant but act hierarchically and synergistically to achieve optimal EC activity. Such exquisite kinetic regulation may reflect the speed-changing events during the transcription cycle, such as pause-release and termination, and enable cells to adapt to a changing environment.

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RNA聚合酶II（Pol II）介导的转录延伸是真核生物基因表达中的关键步骤。虽然已有大量研究表明，Pol II的延伸速度受多种延伸因子的调控，但其具体的调控机制，尤其是在高等真核生物中的调控机制仍未完全被揭示。

本次线上直播讲座，我们有幸邀请到来自美国洛克菲勒大学刘诗欣组的王昱焜博士，分享其在单分子水平重建哺乳动物转录延伸复合物（EC）平台方面的突破性研究成果。该研究利用LUMICKS C-Trap实时观察EC的延伸与停顿过程，揭示各类延伸因子如何协同调控Pol II动力学状态，推动转录过程的精细调节。

Get an inside look at the new C-Trap Accelerater Suite and see how it can transform your experiments. Our product managers will walk you through the powerful new features, which are designed to make your C-Trap faster, easier to use, and more reproducible than ever.

The nuclear envelope protects the genome from mechanical stress during processes such as migration, division, and compression , but how it buffers forces at the scale of DNA remains unclear. Here, we utilize optical tweezers to show that a multivalent protein–DNA co-condensate containing the nuclear envelope protein LEM2 and the DNA-binding protein BAF shield DNA beyond its melting point at 65 pN. Under load, their collective assembly induces an unconventional DNA stiffening effect that provides mechanical reinforcement, dependent on the intrinsically disordered region (IDR) of LEM2. Within cells, these components form an elastic surface hydrogel at the nuclear periphery, visible as a continuous surface by cryo-electron tomography. Disruption of this surface hydrogel increases DNA damage and micronuclei formation during nuclear deformation. Together, this work expands the functional repertoire of condensates, revealing a load-responsive nuclear surface hydrogel at the mesoscale that mitigates mechanical stress.

Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and gastric carcinoma and predict response to FGFR kinase inhibitors. However, response rates and durability are limited due to the emergence of resistance, typically involving FGFR2 kinase domain mutations, and to sub-optimal dosing, relating to adverse drug effects.

This webcast will present new work showing that the vast majority of such alterations retain the extracellular domain (ECD), potentially enabling highly selective targeting of the FGFR2 ECD using biotherapeutics.

To improve on the activity of traditional bivalent monotopic antibodies, the Sellers lab systematically generated biparatopic antibodies targeting distinct epitope pairs in FGFR2 ECD, and identified antibodies that effectively block signaling and malignant growth driven by FGFR2-fusions.

These antibodies robustly blocked proliferation and colony formation in FGFR2-fusion driven cholangiocarcinoma and demonstrated robust in vivo anti-tumour activity. In vivo activity was marked by significant antibody-mediated downregulation of FGFR2 and in turn this was associated with robust lysosomal internalization enacted by the two biparatopics. In vitro, the biparatopic antibodies demonstrated activity against FGFR inhibitor resistant alleles of FGFR2. The internalization properties of the antibodies also make them suitable for exploration as antibody-drug conjugates

Structural Maintenance of Chromosome (SMC) complexes, cohesin, condensin and Smc5/6, play a fundamental role in genome organization, facilitating chromosome compaction, segregation, and DNA repair. Despite their essential functions, the mechanisms by which the complexes interact with different DNA substrates and influence topological transitions remain not fully understood. Using the LUMICKS C-Trap, we have employed single-molecule approaches to analyze the behavior of purified SMC complexes, focusing on yeast cohesin and human SMC5/6, on different DNA substrates, including double-stranded (dsDNA) and single-stranded DNA (ssDNA). Additionally, we have used a quadrupole optical trap to bridging by SMC complexes and their effect on DNA decatenation by Topoisomerase IIα (Top2A). These findings provide new insights into the fundamental properties and requirements of cohesin and Smc5/6’s interaction with DNA substrates, as well as their ability to bridge two independent DNAs.