The nuclear lamina is thought to be the primary mechanical defence of the nucleus. However, the lamina is integrated within a network of lipids, proteins and chromatin; the interdependence of this network poses a challenge to defining the individual mechanical contributions of these components. Here, we isolate the role of chromatin in nuclear mechanics by using a system lacking lamins. Using novel imaging analyses, we observe that untethering chromatin from the inner nuclear membrane results in highly deformable nuclei in vivo, particularly in response to cytoskeletal forces. Using optical tweezers, we find that isolated nuclei lacking inner nuclear membrane tethers are less stiff than wild-type nuclei and exhibit increased chromatin flow, particularly in frequency ranges that recapitulate the kinetics of cytoskeletal dynamics. We suggest that modulating chromatin flow can define both transient and long-lived changes in nuclear shape that are biologically important and may be altered in disease.

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Although convolutional neural networks (CNNs) have been applied to a variety of computational genomics problems, there remains a large gap in our understanding of how they build representations of regulatory genomic sequences. Here we perform systematic experiments on synthetic sequences to reveal how CNN architecture, specifically convolutional filter size and max-pooling, influences the extent that sequence motif representations are learned by first layer filters. We find that CNNs designed to foster hierarchical representation learning of sequence motifs-assembling partial features into whole features in deeper layers-tend to learn distributed representations, i.e. partial motifs. On the other hand, CNNs that are designed to limit the ability to hierarchically build sequence motif representations in deeper layers tend to learn more interpretable localist representations, i.e. whole motifs. We then validate that this representation learning principle established from synthetic sequences generalizes to in vivo sequences.

Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the membrane-binding proteins islet amyloid popypeptide (IAPP) and α-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and also facilitates accurate solution-based measurements. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required.

Cytochrome P450 3A4 is a major human drug-metabolizing enzyme, and displays pharmacologically-relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector α-naphthoflavone. We find direct evidence that CYP3A4 effectors can decrease substrate off-rates, providing a possible mechanism for effector-mediated enhancement of substrate metabolism. These interesting results highlight the potential of SM methods in studies of CYP allosteric mechanisms.Cytochrome P450 (CYP) isoform 3A4 (CYP3A4) is the predominant human drugmetabolizing enzyme, responsible for metabolizing ∼50% of commercial pharmaceuticals. 1 CYP3A4 displays a wide variety of allosteric (non-hyperbolic) kinetic behavior that may cause atypical CYP3A4-mediated pharmacokinetics. CYP allosterism results from the simultaneous binding of multiple substrate and/or effector molecules. 2-9 Here, in the first single-molecule (SM) fluorescence studies of CYPs, we measure residence times of the fluorescent dye Nile Red (NR) bound to CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, 10, 11 with and without the effector α-naphthoflavone (ANF). We find good agreement with ensemble kinetic measurements, as well as direct evidence that CYP3A4 effectors can modulate substrate off-rates. These results highlight the utility of SM methods in studies of CYP allosteric mechanisms.In recent work, we described NR as a reporter substrate of allosteric behavior in CYP3A4: 12, 13 NR forms spectrally distinct singly-and doubly-occupied complexes with CYP3A4 (K D s of 0.3 μM and 2.2 μM respectively). The second binding induces a ∼8-fold greater heme spin winky@u.washington.edu. shift than the first. NR fluorescence also responds to changes in the active-site environment induced by ANF bound to a separate site. 8 ANF raises the k cat for NR by ∼3-fold (C. Fernandez and J. Lampe, unpublished data). NIH Public AccessHere, we extend these studies to CYP3A4 in Nanodiscs. Nanodiscs consist of a lipid bilayer ∼10 nm in diameter stabilized by a helical protein coat, and provide a biologically relevant membrane environment for CYP3A4. SM kinetic studies of CYP3A4-Nanodiscs require a detailed thermodynamic understanding of the multiple possible binding modes. The Nanodisc bilayer may compete with CYP3A4 for NR binding; moreover, ligands can bind to CYP3A4-Nanodiscs with significantly different affinity than to recombinant protein in solution. 14 To extend the previous equilibrium binding analysis to NR and aid in interpretation of SM measurements, we combined steady-state fluorescence spectroscopy and UV-Vis spectroscopy of NR binding Nanodiscs and CYP3A4-Nanodiscs.NR displays significant fluorescence enhancement relative to buffer upon binding either CYP3A4 or the Nanodisc membrane. N...

Deep convolutional neural networks (CNNs) trained on regulatory genomic sequences tend to learn distributed feature representations across many filters, making it challenging to decipher biologically meaningful representations, such as sequence motifs. Here we perform a comprehensive analysis on synthetic sequences to investigate the role that CNN activations have on model interpretability. We introduce a novel application of the exponential activation that when applied to first layer filters, consistently leads to interpretable and robust representations of motifs compared to other commonly used activations, both qualitatively and quantitatively. Strikingly, we demonstrate that CNNs with better test performance do not necessarily imply more interpretable representations with attribution methods. We find that CNNs with exponential activations significantly improve the efficacy of a CNN's ability to recover biologically meaningful representations with attribution methods. We demonstrate these results generalize to real DNA sequences across several in vivo datasets. Together, this work demonstrates how a small modification to existing CNNs, i.e. setting exponential activations in the first layer, can significantly improve the robustness and interpretabilty of learned representations directly in convolutional filters and indirectly with attribution methods. 3/18 4/18

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Lineage plasticity is a prominent feature of pancreatic ductal adenocarcinoma (PDA) cells, which can occur via deregulation of lineage-specifying transcription factors. Here, we show that the zinc finger protein ZBED2 is aberrantly expressed in PDA and alters tumor cell identity in this disease. Unexpectedly, our epigenomic experiments reveal that ZBED2 is a sequence-specific transcriptional repressor of IFN-stimulated genes, which occurs through antagonism of IFN regulatory factor 1 (IRF1)-mediated transcriptional activation at cooccupied promoter elements. Consequently, ZBED2 attenuates the transcriptional output and growth arrest phenotypes downstream of IFN signaling in multiple PDA cell line models. We also found that ZBED2 is preferentially expressed in the squamous molecular subtype of human PDA, in association with inferior patient survival outcomes. Consistent with this observation, we show that ZBED2 can repress the pancreatic progenitor transcriptional program, enhance motility, and promote invasion in PDA cells. Collectively, our findings suggest that high ZBED2 expression is acquired during PDA progression to suppress the IFN response pathway and to promote lineage plasticity in this disease.