Genome-wide epigenomic maps have revealed millions of putative enhancers and promoters, but experimental validation of their function and high-resolution dissection of their driver nucleotides remain limited. Here, we present HiDRA (High-resolution Dissection of Regulatory Activity), a combined experimental and computational method for high-resolution genome-wide testing and dissection of putative regulatory regions. We test ~7 million accessible DNA fragments in a single experiment, by coupling accessible chromatin extraction with self-transcribing episomal reporters (ATAC-STARR-seq). By design, fragments are highly overlapping in densely-sampled accessible regions, enabling us to pinpoint driver regulatory nucleotides by exploiting differences in activity between partially-overlapping fragments using a machine learning model (SHARPR-RE). In GM12878 lymphoblastoid cells, we find ~65,000 regions showing enhancer function, and pinpoint ~13,000 high-resolution driver elements. These are enriched for regulatory motifs, evolutionarily-conserved nucleotides, and disease-associated genetic variants from genome-wide association studies. Overall, HiDRA provides a high-throughput, high-resolution approach for dissecting regulatory regions and driver nucleotides.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinasedependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src͞abl inhibitor BMS-354825 and exposed the expected side activities for Glivec͞STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.drug discovery ͉ high-throughput screening ͉ tyrosine kinase T he ability to simultaneously interrogate the activities of a library of molecules against a large panel of cellular assays would provide a rapid efficient means to begin to characterize and correlate the biological properties of both natural and synthetic chemical diversity. For example, libraries of noncoding RNAs, mutant growth factors, small molecule kinase inhibitors, or even existing drugs could be assayed for their potency and selectivity in pathway-based or receptor screens or toxicity and metabolic stability in diverse cell types to discover a new biological activity or optimize the pharmacological properties of a molecule (1-3). Although whole-cell systems represent an attractive milieu to characterize gene and small-molecule function, no robust and systematic method exists to correlate chemical structure and biological activity across a large number of molecules and cellular assays. To address this problem, we have developed an approach that affords rapid cost-effective broad-based cellular profiling in parallel against molecular libraries. An industrial-scale automated compound profiling (ACP) system has been designed, which consists of an automated tissue culture system for propagating cell lines, integrated with a system for automatically performing miniaturized cell-based assays in 384-or 1,536-well microplates. The ACP can rapidly test thousands of arrayed molecules, in replicates, in doseresponse format against hundreds of unique cellular assays in a single experiment.To demonstrate this capability, we focused on the problem of identifying selective small-molecule inhibitors of protein tyrosine kinases. Tyrosine ...

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.tandem repeat | high strength | protein | thermoplastic | squid ring teeth P roteins are heteropolymers that provide a variety of building blocks for designing biological materials (1). Proteins have several advantages as natural materials: (i) their chain length, sequence, and stereochemistry can be easily controlled, (ii) the molecular structure of proteins is well-defined (e.g., secondary, tertiary, and quaternary structures), (iii) they provide a variety of functional chemistries for conjugation to other biomolecules or polymers, and (iv) they can be designed to exhibit a variety of physical properties (2). Proteins are diverse but often display substantial similarity in sequence and 3D structure. Duplication of structural units is a natural evolutionary strategy for increasing the complexity of both globular and fibrous/ structural proteins (3). For example, collagen has polyproline-and glycine-rich helices, whereas silk and elastin have β-spiral [GPGXX], linker [GP(S,Y,G)], and 3 10 -helix [GGX] repeats. These repetitions are advantageous because of the intrinsic promotion of stability through the periodic recurrence of favorable interactions (4-7).A new family of repetitive structural proteins was recently identified in the tentacles of several squid species (8, 9). Squid have teeth-like structures inside their suckers that allow the animals to grip tightly on a diverse array of objects (10). Using the tools of molecular biology and proteomics, it has been shown that these squid ring teeth (SRT) proteins have segmented semicrystalline morphology with repetitive amorphous and crystalline domains. SRT-based materials were shown to have high elastic modulus: 4-8 GPa in air and 2-4 GPa underwater below the glass transition temperature (11). However, a clear relationship between the molecular structure and the mechanical properties of this material remains elusive. This problem is complex, because SRT proteins are polydispersed i...

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Abstractselected from accessible chromatin in the GM12878 lymphoblastoid cell line. By design, accessibilityselected fragments were highly overlapping (up to 370 per region), enabling us to pinpoint driver regulatory nucleotides by exploiting subtle differences in reporter activity between partially-overlapping fragments, using a new machine learning model SHARPR2. Our resulting maps include ~65,000 regions showing significant enhancer function and enriched for endogenous active histone marks (including H3K9ac, H3K27ac), regulatory sequence motifs, and regions bound by immune regulators. Within them, we discover ~13,000 high-resolution driver elements enriched for regulatory motifs and evolutionarily-conserved nucleotides, and help predict causal genetic variants underlying disease from genome-wide association studies. Overall, HiDRA provides a general, scalable, high-throughput, and high-resolution approach for experimental dissection of regulatory regions and driver nucleotides in the context of human biology and disease.peer-reviewed)