Detecting hierarchical genome folding with network modularity.

Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Here, we describe 3DNetMod, a graph theory-based method for sensitive and accurate detection of chromatin domains across length scales in Hi-C data. We identify nested, partially overlapping TADs and subTADs genome wide by optimizing network modularity and varying a single resolution parameter. 3DNetMod can be applied broadly to understand genome reconfiguration in development and disease.

Crossed wires: 3D genome misfolding in human disease.

Mammalian genomes are folded into unique topological structures that undergo precise spatiotemporal restructuring during healthy development. Here, we highlight recent advances in our understanding of how the genome folds inside the 3D nucleus and how these folding patterns are miswired during the onset and progression of mammalian disease states. We discuss potential mechanisms underlying the link among genome misfolding, genome dysregulation, and aberrant cellular phenotypes. We also discuss cases in which the endogenous 3D genome configurations in healthy cells might be particularly susceptible to mutation or translocation. Together, these data support an emerging model in which genome folding and misfolding is critically linked to the onset and progression of a broad range of human diseases.

Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming.

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.

Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.

BACKGROUND: Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational loop-grafting approach to engineer AgTx variants that bound to αvß3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins. CONCLUSIONS/SIGNIFICANCE: In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.