Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.

Broad epitope coverage of a human in vitro antibody library.

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

A genome-wide 3C-method for characterizing the three-dimensional architectures of genomes.

Accumulating evidence demonstrates that the three-dimensional (3D) organization of chromosomes within the eukaryotic nucleus reflects and influences genomic activities, including transcription, DNA replication, recombination and DNA repair. In order to uncover structure-function relationships, it is necessary first to understand the principles underlying the folding and the 3D arrangement of chromosomes. Chromosome conformation capture (3C) provides a powerful tool for detecting interactions within and between chromosomes. A high throughput derivative of 3C, chromosome conformation capture on chip (4C), executes a genome-wide interrogation of interaction partners for a given locus. We recently developed a new method, a derivative of 3C and 4C, which, similar to Hi-C, is capable of comprehensively identifying long-range chromosome interactions throughout a genome in an unbiased fashion. Hence, our method can be applied to decipher the 3D architectures of genomes. Here, we provide a detailed protocol for this method.

A three-dimensional model of the yeast genome.

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or 'factories' for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Capture and sequence analysis of RNAs with terminal 2',3'-cyclic phosphates.

The combination of ligation-based RNA capture methods and high-throughput sequencing has facilitated the characterization of transcriptomes and the identification of novel noncoding RNAs. However, current ligation-based RNA capture methods require RNA substrates with terminal 3'-hydroxyl groups, limiting their utility for identifying RNAs with modified termini like 2',3'-cyclic phosphates. Cyclic phosphate-terminated RNAs are generated by endonucleolytic cleavages and self-cleaving ribozymes and are found as stable modifications on cellular RNAs such as the U6 spliceosomal RNA. We developed a method that uses the Arabidopsis thaliana tRNA ligase to add an adaptor oligonucleotide to RNAs that terminate in 2',3'-cyclic phosphates. The adaptor allows specific priming by reverse transcriptase, which is followed by additional steps for PCR amplification and high-throughput DNA sequencing. Applying the method to total human RNA, we found 2836 sequencing reads corresponding to the 3' terminus of U6 snRNA, validating the method. In addition to a large background of reads that map throughout abundantly transcribed RNAs, we also found 42,324 reads of specific fragments from several tRNA isoacceptor families, suggesting that this method may identify processing events previously undetected by other RNA cloning techniques.

A WAVE-1 and WRP signaling complex regulates spine density, synaptic plasticity, and memory.

The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.