Biology subjects

Insigne, K. D.

Publications and source records attributed to Insigne, K. D..

3 recordsLinked to original sources

bioRxiv2020

Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8,269 rationally designed IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. Using these data, we fit a statistical mechanics model that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three novel promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.

synthetic biology↗

bioRxiv2019

Multiplexed dissection of a model human transcription factor binding site architecture

In eukaryotes, transcription factors orchestrate gene expression by binding to TF-Binding Sites (TFBSs) and localizing transcriptional co-regulators and RNA Polymerase II to cis-regulatory elements. The strength and regulation of transcription can be modulated by a variety of factors including TFBS composition, TFBS affinity and number, distance between TFBSs, distance of TFBSs to transcription start sites, and epigenetic modifications. We still lack a basic comprehension of how such variables shaping cis-regulatory architecture culminate in quantitative transcriptional responses. Here we explored how such factors determine the transcriptional activity of a model transcription factor, the c-AMP Response Element (CRE) binding protein. We measured expression driven by 4,602 synthetic regulatory elements in a massively parallel reporter assay (MPRA) exploring the impact of CRE number, affinity, distance to the promoter, and spacing between multiple CREs. We found the number and affinity of CREs within regulatory elements largely determines overall expression, and this relationship is shaped by the proximity of each CRE to the downstream promoter. In addition, while we observed expression periodicity as the CRE distance to the promoter varied, the spacing between multiple CREs altered this periodicity. Finally, we compare library expression between an episomal MPRA and a new, genomically-integrated MPRA in which a single synthetic regulatory element is present per cell at a defined locus. We observe that these largely recapitulate each other although weaker, non-canonical CREs exhibited greater activity in the genomic context.

genomics↗

bioRxiv2017

Large-scale screening of rare genetic variants in humans reveals frequent splicing disruptions

Any individuals genome contains [~]4-5 million genetic variants that differ from reference, and understanding how these variants give rise to trait diversity and disease susceptibility is a central goal of human genetics1. A vast majority (96-99%) of an individuals variants are common, though at a population level the overwhelming majority of variants are rare2-5. Because of their scarcity in an individuals genome, rare variants that play important roles in complex traits are likely to have large functional effects6,7. Mutations that cause an exon to be skipped can have severe functional consequences on gene function, and many known disease-causing mutations reduce or eliminate exon recognition8. Here we explore the extent to which rare genetic variation in humans results in near complete loss of exon recognition. We developed a Multiplexed Functional Assay of Splicing using Sort-seq (MFASS) that allows us to measure exon inclusion in thousands of human exons and surrounding intronic sequence simultaneously. We assayed 27,733 extant variants in the Exome Aggregation Consortium (ExAC)9 within or adjacent to 2,339 human exons, and found that 3.8% (1,050) of the variants, almost all of which were extremely rare, led to large-effect defects in exon recognition. Importantly, we find that 83% of these splice-disrupting variants (SDVs) are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate that loss of exon recognition is an important and underappreciated means by which rare variants exert large functional effects, and that MFASS enables their empirical assessment for splicing defects at scale.

genetics↗