Optical control of mammalian endogenous transcription and epigenetic states

Abstract

The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

Figure 1. Design and Optimization of the LITE system

(a) Schematic of the LITE system. Light stimulation induces dimerization of CRY2 and CIB1, recruiting the effector to the target promoter. (b) LITE architecture was optimized by fusing TALE and the transcriptional activator VP64^, to different truncations of CRY2 and CIB1 (n next to each bar). (c) Time course of light-dependent Neurog2 up-regulation and decay post-illumination (n = 3 biological replicas; *, p < 0.05; ***, p < 0.001). Cells were stimulated with 5 mW/cm² light (460nm, 1 s pulses at 0.066 Hz). Mean ± s.e.m in all panels.

Figure 2. In vitro and in vivo AAV-mediated TALE delivery targeting endogenous loci in neurons

(a) Schematic AAV vectors for TALE delivery. (b) Representative images of primary cortical neurons expressing TALE-VP64. (c) TALE-VP64 constructs targeting a variety of endogenous neuronal genes were screened for transcriptional activation in primary cortical neurons (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 3 biological replicas). (d) TALE-VP64 expression in PFC. (e) Higher magnification image of TALE-VP64-expressing neurons in PFC. (f) Grm2 mRNA up-regulation by TALE-VP64 in vivo in PFC (n = 4 animals). Mean ± s.e.m in all panels.

Figure 3. LITE-mediated optogenetic modulation of endogenous transcription in primary neurons and in vivo

(a) Schematic of AAV-LITE constructs. (b) Images of primary neurons expressing LITE constructs. (c) Light-induced activation of Grm2 in primary neurons after 24 h of stimulation (250 ms pulses at 0.033Hz or 500 ms pulses at 0.016Hz; 5mW/cm²; n = 4 biological replicas). (d) Up-regulation of Grm2 in primary cortical neurons after 4 h or 24 h of stimulation. Expression levels are shown relative to neurons transduced with GFP only (number of biological replica denoted within graph bars). (e) Light-mediated changes in mGluR2 protein levels (n = 7 biological replica). (f) Schematic of in vivo optogenetic stimulation setup. (g) Representative images of PFC neurons expressing both LITE components. (h) Light-induced activation of endogenous Grm2 expression using LITEs transduced into ILC. (**, p < 0.05; number of animals denoted within graph bars) (i) LITE2.0 significantly reduces the level of background activation in Neuro 2a cells (n = 3 biological replica). Mean ± s.e.m in all panels.

Figure 4. TALE- and LITE-mediated epigenetic modifications

(a) LITE epigenetic modifiers (epiLITE). (b) epiLITE AAV vectors. (c) epiLITE-mediated repression of endogenous Grm2 in neurons (n = 4 biological replicas). (d) epiLITE-mediated decrease in H3K9 histone acetylation at the Grm2 promoter (n = 4 biological replicas). (e, f) epiTALE-methyltransferases mediated decrease in Grm2 mRNA and corresponding enrichment of H3K9me1, H4K20me3, and H3K27me3 at the Grm2 promoter (n denoted within graph). (g, h) epiTALE histone deacetylases mediated repression of Grm2 and corresponding decreases in H4K8Ac and H3K9Ac marks at the Grm2 promoter (n denoted within graph). Mean ± s.e.m in all panels.