Single molecule imaging of Ascl1 during neuronal reprogramming - 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.
