We propose to develop and apply a paradigm-shifting technological platform that uses a series of Light- Inducible Transcriptional Effectors (LITEs) to orchestrate the temporal regulation of multiple genes in both individual cells in vitro and in the intact organism. The technology we propose to develop will be very broadly applicable and has the potential to radically transform the scale and rate of discovery across different biomedical fields. Application of this novel technology will enable high throughput discovery of the upstream transcriptional regulatory elements of any endogenous gene, as well as temporally precise modulation of gene expression in the native genome. Precise temporal and spatial patterns of gene expression are observed in different tissue and cell types, and are orchestrated and maintained by complex transcriptionally regulated circuits involving multiple genes. Due to the lack of integrated control and readout technologies that enable simultaneous perturbation and "fast" tuning of multiple genes, our ability to causally link transcriptional network dynamics with physiology and development remains at the infancy stage. The LITEs platform will enable modification and regulation of gene expression on the time-scale of hours using a non-invasive, light-mediated inductive strategy, thereby enabling a new generation of interactive genetic studies currently inaccessible with conventional techniques. Our proposal consists of two main components: 1) Novel technology development of light sensitive designer zinc finger (ZF) transcriptional modulators (LITEs-ZF). Genomic, synthetic biology, and protein engineering approaches will be used to develop a suite of novel light-inducible transcriptional regulators targeted at specific genes in the native genome of mammalian cells. Since ZF DNA binding domains can be engineered to target any DNA sequence, ZF-LITEs are applicable to a broad range of biological research studies in a variety of different organisms and cells types. 2) Application of the technology toward high-throughput in vitro and in vivo interrogation of transcriptional network dynamics in the central nervous system. In vitro application will be aimed at identification of upstream effectors of gene expression critical to the differentiation of corticospinal motor neurons (CSMN), a clinically relevant neuronal population that degenerates in amyotrophic lateral sclerosis (ALS) and is injured in spinal cord injury. In vivo application will be focused on directing the regeneration of CSMN by mimicking specific temporal sequences of CSMN-specific developmental cues within the adult brain. Given the broad applicability of this technology, the impact of this proposed work will be far reaching and will radically transform existing experimental approaches for studying gene interactions in all fields of life science and medicine.

Public Health Relevance

Understanding the transcriptional networks that drive cellular development and repair has landscape-shifting impacts in the field of regenerative medicine. Here we develop a non-invasive, optical technology to enable genome-wide tuning of gene expression dynamics toward regeneration of corticospinal motor neurons.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS073124-05
Application #
8470731
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Mamounas, Laura
Project Start
2010-09-27
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
5
Fiscal Year
2013
Total Cost
$629,595
Indirect Cost
$100,145
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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