3D genome, on repeat: Higher-order folding principles of the heterochromatinized repetitive genome.

Nearly half of the human genome is comprised of diverse repetitive sequences ranging from satellite repeats to retrotransposable elements. Such sequences are susceptible to stepwise expansions, duplications, inversions, and recombination events which can compromise genome function. In this review, we discuss the higher-order folding mechanisms of compartmentalization and loop extrusion and how they shape, and are shaped by, heterochromatin. Using primarily mammalian model systems, we contrast mechanisms governing H3K9me3-mediated heterochromatinization of the repetitive genome and highlight emerging links between repetitive elements and chromatin folding.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Cohesin-mediated loop anchors confine the locations of human replication origins.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

Rapid sensing of clinically relevant glutamine concentrations in human serum with metabolically engineered E. coli-based cell-free protein synthesis.

Bioavailable glutamine (Gln) is critical for metabolism, intestinal health, immune function, and cell signaling. Routine measurement of serum Gln concentrations could facilitate improved diagnosis and treatment of severe infections, anorexia nervosa, chronic kidney disease, diabetes, and cancer. Current methods for quantifying tissue Gln concentrations rely mainly on HPLC, which requires extensive sample preparation and expensive equipment. Consequently, patient Gln levels may be clinically underutilized. Cell-free protein synthesis (CFPS) is an emerging sensing platform with promising clinical applications, including detection of hormones, amino acids, nucleic acids, and other biomarkers. In this work, in vitro E. coli amino acid metabolism is engineered with methionine sulfoximine to inhibit glutamine synthetase and create a CFPS Gln sensor. The sensor features a strong signal-to-noise ratio and a detection range ideally suited to physiological Gln concentrations. Furthermore, it quantifies Gln concentration in the presence of human serum. This work demonstrates that CFPS reactions which harness the metabolic power of E. coli lysate may be engineered to detect clinically relevant analytes in human samples. This approach could lead to transformative point-of-care diagnostics and improved treatment regimens for a variety of diseases including cancer, diabetes, anorexia nervosa, chronic kidney disease, and severe infections.

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport.

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized "cell-free" protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These "hydrofoam" reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are "cell-free" and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.

Engineering Cell-Free Protein Synthesis for High-Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On-Demand Treatment of Acute Lymphoblastic Leukemia.

Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell-free protein synthesis (CFPS) of FDA approved l-asparaginase (crisantaspase) is demonstrated by employing an aspartate-fed-batch reactor format. A CFPS-based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf-stable and low-cost Escherichia coli-based CFPS reactions may be employed on-demand to 1) synthesize biologics on-site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not-too-distant future when shelf-stable E. coli CFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.