Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Context-dependent neocentromere activity in synthetic yeast chromosome VIII.

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.