The ability to directly convert differentiated somatic cell types into neuronal cells provides critical information about what defines a neuron. The methods of direct neuronal reprogramming have been greatly informed by neurodevelopment, and through examining this synthetic scenario, insights into neurodevelopment have reciprocally been discovered. This fundamental knowledge of neuronal identity is necessary in order to better understand the deficits that occur in neuropsychiatric disorders such as autism and schizophrenia. Additionally, elucidation of the mechanisms underlying direct conversion have the potential to create more comprehensive models of neurological disorders with which to study disease biology and perform drug screening, as well as possibilities for more efficient regenerative medicine methods. It has been shown that the expression of three neuronal transcription factors, Ascl1, Brn2 and Myt1l, is able to directly convert mouse embryonic fibroblasts (MEFs) into functional induced neuronal (iN) cells. Ascl1 appears to be a major driver of this phenomenon, as expression of Ascl1 alone is able to produce iN cells, although at a lower efficiency. Ascl1 is a proneural basic helix loop helix (bHLH) transcription factor that is expressed endogenously in neural precursor cells (NPCs), where its expression oscillates to maintain progenitor status. When its expression becomes high and sustained, the cells differentiate into neurons. In reprogramming, Ascl1 appears to have a unique ability among other reprogramming factors to find and locates its precise cognate binding sites not only in its endogenous context of NPCs, but also in the different chromatin context of fibroblasts. This feature led to Ascl1 being described as an ?on target? pioneer factor, as it can bind its targets even when they are found in closed chromatin. While it is known that Ascl1 can activate its endogenous targets in different cell types, it is unclear how precisely Ascl1 is able to search for and locate its targets in regions of closed chromatin. Recent advances in single molecule imaging provide a great increase in the spatial and temporal resolution of tracking transcription factors within the nucleus, and it is now possible to analyze individual molecules and their dynamic interactions with DNA. This proposal aims to utilize single molecule imaging techniques to understand how Ascl1 can activate its targets in the different epigenetic contexts of neurons and fibroblasts. Using highly inclined and laminated optical sheet (HILO) microscopy, the binding kinetics and search patterns of Ascl1 in its physiological context of NPCs will be characterized and compared to the scenario when Ascl1 is overexpressed in fibroblasts undergoing reprogramming. These characteristics will also be measured in areas of the nucleus labeled as heterochromatin and compared to more accessible regions, to determine if Ascl1's ability to locate its proper targets is due to a unique search pattern in regions of closed chromatin. Future directions will aim to investigate the dynamic interactions of Ascl1 with the 3D chromatin architecture to coordinately regulate individual genes within the gene expression network that orchestrates reprogramming.

Public Health Relevance

Understanding how fibroblasts are directly converted to induced neuronal (iN) cells can produce better models of complex neurological disorders as well as fundamental insights into neuronal lineage specification. This work proposes to use single molecule imaging to elucidate how Ascl1 dynamically acts within the nucleus to orchestrate this conversion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32NS105321-02
Application #
9619979
Study Section
Special Emphasis Panel (ZNS1)
Program Officer
Lavaute, Timothy M
Project Start
2017-12-01
Project End
2020-03-31
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Stanford University
Department
Pathology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304