Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a "pincer-like" conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitinylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

H2A.Z deposition by SWR1C involves multiple ATP-dependent steps.

Histone variant H2A.Z is a conserved feature of nucleosomes flanking protein-coding genes. Deposition of H2A.Z requires ATP-dependent replacement of nucleosomal H2A by a chromatin remodeler related to the multi-subunit enzyme, yeast SWR1C. How these enzymes use ATP to promote this nucleosome editing reaction remains unclear. Here we use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction. Real-time analysis of single nucleosome remodeling events reveals an initial priming step that occurs after ATP addition that involves a combination of both transient DNA unwrapping from the nucleosome and histone octamer deformations. Priming is followed by rapid loss of histone H2A, which is subsequently released from the H2A.Z nucleosomal product. Surprisingly, rates of both priming and the release of the H2A/H2B dimer are sensitive to ATP concentration. This complex reaction pathway provides multiple opportunities to regulate timely and accurate deposition of H2A.Z at key genomic locations.

Emergent laparoscopic surgical intervention for perforated hemorrhagic cholecystitis with hemodynamic instability.

Hemorrhagic cholecystitis is a rare diagnosis that closely mimics acute cholecystitis. Physical examination, laboratory studies and, in particular, computed tomography imaging allow for rapid diagnosis, stabilization and emergent surgical intervention. We describe our experience with three patients requiring emergent surgical intervention for hemorrhagic cholecystitis with unique clinical features including decreased platelet function due to liver cirrhosis, dual antiplatelet therapy and intraoperative finding of cholecystohepatic communication. Furthermore, we provide video recordings of two cases highlighting the severity of the disease. All presented patients were hemodynamically unstable and showed peritoneal signs on exam. Laboratory studies revealed moderate anemia and leukocytosis, while computed tomography suggested hemorrhage in the gallbladder. All patients required blood transfusions during their care and underwent laparoscopic cholecystectomy. Hemoperitoneum and gallbladder perforation were confirmed intraoperatively. Patients fully recovered without significant postoperative complications due to expedited operative management.

Fluorescence approaches for biochemical analysis of ATP-dependent chromatin remodeling enzymes.

The dynamic nature of chromatin is an essential mechanism by which gene expression is regulated. Chromatin is comprised of nucleosomes, an octamer of histone proteins wrapped by DNA, and manipulation of these structures is carried out by a family of proteins known as ATP-dependent chromatin remodeling enzymes. These enzymes carry out a diverse range of activities, from appropriately positioning and adjusting the density of nucleosomes on genes, to installation and removal of histones for sequence variants, to ejection from DNA. These activities have a critical role in the proper maintenance of chromatin architecture, and dysregulation of chromatin remodeling is directly linked to the pathophysiology of various diseases. Mechanistic understanding of chromatin remodeling enzymes is therefore desirable, both as the drivers of this essential cellular activity and as potentially novel therapeutic targets in disease. In this chapter we cover our current methods for characterization of remodeler substrate binding affinity and catalytic activity, leveraging fluorescence polarization and Förster resonance energy transfer assays.

A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7.

The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3' ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.

Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.