Amplification of chromosomal region 8p11-12 is a frequent genetic alteration implicated in the etiology of lung squamous cell carcinoma (LUSC) 1 - 3 . FGFR1 (fibroblast growth factor receptor 1) is the main candidate driver within this region 4 . However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful 5 . Here we identify the H3K36 methyltransferase NSD3 ( n uclear receptor binding S ET d omain protein 3 ), an 8p11-12-localized gene, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased NSD3 expression strongly correlates with its gene amplification. Ablation of NSD3 , but not FGFR1 , attenuates tumor growth and extends survival in a potent LUSC mouse model. We identify NSD3 T1232A as an LUSC-associated variant that increases H3K36 dimethylation (H3K36me2) catalytic activity in vitro and in vivo . Structural dynamic analyses reveal that the T1232A substitution elicits localized mobility changes throughout NSD3’s catalytic domain to relieve auto-inhibition and increase H3 substrate accessibility. NSD3 T1232A expression in vivo accelerates tumorigenesis and decreases overall survival in LUSC mouse models. Pathologic H3K36me2 generation by NSD3 T1232A rewires the chromatin landscape to promote oncogenic gene expression programming. Further, NSD3’s catalytic activity promotes human tracheobronchial cell transformation and xenograft growth of human 8p11-12-amplified LUSC cell lines. NSD3 depletion in patient-derived xenografts (PDXs) from primary LUSC harboring NSD3 amplification or the NSD3 T1232A variant attenuates neoplastic growth. Finally, NSD3-regulated LUSC PDXs are markedly sensitive to bromodomain inhibition (BETi). Together, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to BETi.

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The DNA-binding protein H-NS is a pleiotropic gene regulator in gram-negative bacteria. Through its capacity to sense temperature and other environmental factors, H-NS allows pathogens like Salmonella to adapt their gene expression to their presence inside or outside warm-blooded hosts. To investigate how this sensing mechanism may have evolved to fit different bacterial lifestyles, we compared H-NS orthologs from bacteria that infect humans, plants, and insects, and from bacteria that live on a deep-sea hypothermal vent. The combination of biophysical characterization, high-resolution proton-less nuclear magnetic resonance spectroscopy, and molecular simulations revealed, at an atomistic level, how the same general mechanism was adapted to specific habitats and lifestyles. In particular, we demonstrate how environment-sensing characteristics arise from specifically positioned intra- or intermolecular electrostatic interactions. Our integrative approach clarified the exact modus operandi for H-NS-mediated environmental sensing and suggested that this sensing mechanism resulted from the exaptation of an ancestral protein feature.

280 different patterns of histone modifications were installed in preassembled nucleosomes using PTS and SML enabling screening of readout crosstalk.

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15N{1H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

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Efficient determination of protein ligandability, or the propensity to bind small-molecules, would greatly facilitate drug development for novel targets. Ligandability is currently assessed using computational methods that typically consider the static structural properties of putative binding sites or by experimental fragment screening. Here, we evaluate ligandability of conserved BTB domains from the cancer-relevant proteins LRF, KAISO, and MIZ1. Using fragment screening, we discover that MIZ1 binds multiple ligands. However, no ligands are uncovered for the structurally related KAISO or LRF. To understand the principles governing ligand-binding by BTB domains, we perform comprehensive NMR-based dynamics studies and find that only the MIZ1 BTB domain exhibits backbone µs-ms time scale motions. Interestingly, residues with elevated dynamics correspond to the binding site of fragment hits and recently defined HUWE1 interaction site. Our data argue that examining protein dynamics using NMR can contribute to identification of cryptic binding sites, and may support prediction of the ligandability of novel challenging targets.

The DNA-binding protein H-NS is a pleiotropic gene regulator in gram-negative bacteria. Through its capacity to sense temperature and other environmental factors, H-NS allows pathogens like Salmonella to adapt their gene expression, and hence toxicity and biological responses, to their presence inside or outside warm-blooded hosts. To investigate how this sensing mechanism may have evolved to fit different bacterial lifestyles, we compared H-NS orthologs from bacteria that infect humans, plants, and insects, and from bacteria that live on a deep-sea hypothermal vent. The combination of biophysical characterization, high-resolution proton-less NMR spectroscopy and molecular simulations revealed, at an atomistic level, how the same general mechanism was adapted to specific habitats and lifestyles. In particular, we demonstrate how environment-sensing characteristics arise from specifically positioned intra-or intermolecular electrostatic interactions. Our integrative approach clarified the mechanism for H-NS-mediated environmental sensing and suggests that it resulted from the exaptation of an ancestral protein feature.