Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2

Abstract

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

Light Control of the Tet Gene Expression System in Mammalian Cells

“…Most light-inducible gene expression systems were established in yeast cells. Some light-inducible gene expression systems optimized in yeast cells do not work efficiently in mammalian cells (Pathak et al, 2017;unpublished data). Therefore, we conducted functional screenings of candi-date constructs in the immortalized human embryonic kidney cell line HEK293T (Figures 1, S1, and S2; Tables S1, S2, S3, S4, and S5).…”

“…However, we did not observe apparent light-induced gene expression in either case when CIB1 and its derivatives were fused to the N terminus or C terminus of p65 AD (Figures S1A-S1D; Tables S1 and S2). This may be due to the nuclear clearing phenotype of Cry2-tethered proteins (Pathak et al, 2017). When Cry2 is fused to certain nuclear-localized proteins having internal dimerization domains, the fused proteins are redistributed outside the nuclei in a lightdependent manner.…”

“…When Cry2 is fused to certain nuclear-localized proteins having internal dimerization domains, the fused proteins are redistributed outside the nuclei in a lightdependent manner. Some Cry2-fused transcription factors undergo substantial nuclear protein clearing upon illumination, resulting in light-dependent loss of protein function (Pathak et al, 2017). It was reported that the nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within Cry2-fused proteins, and TetR has an inherent dimerization domain.…”

“…Optogenetic tools that can control cellular gene expression have been developed, which have the potential to overcome the technical limitations of conventional chemically regulated gene expression systems (Crefcoeur et al, 2013;Hallett et al, 2016;Hö rner et al, 2017;Imayoshi et al, 2013;Konermann et al, 2013;Liu et al, 2012a;Motta-Mena et al, 2014;M€ uller et al, 2013b;Pathak et al, 2017;Polstein and Gersbach, 2012;Shimizu-Sato et al, 2002;Wang et al, 2012;Yazawa et al, 2009). Rapid and dynamic changes of gene expression patterns (e.g., oscillatory changes or stepwise increases) are difficult to address using classical gene expression regulation methods, such as chemically regulated systems, due to their poor temporal and highly invasive characteristics.…”

Chemical physics in living cells — Using light to visualize and control intracellular signal transduction

“…It was observed that certain synthetic transcription factors fused to CRY2 exhibited a clusteringdependent slow nuclear clearance and consequently exhibited diminished TF activity upon blue light illumination. Bidirectional control over gene expression was achieved [171] by coupling this light-dependent transcriptional disruption to light-dependent degradation of its transcriptional target protein [172].…”