Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Dr. R. Jane Rylett

Abstract

The three-dimensional structure of chromatin is essential for context-dependent regulation of gene expression in post-mitotic neurons. Chromosomal rearrangements have been observed in the aging brain, and proteins involved in chromatin organization have altered expression and/or localization in Alzheimer’s disease (AD). A human- and primate-specific transcript of choline acetyltransferase produces an 82-kDa protein (82-kDa ChAT) that is localized to the nucleus of cholinergic neurons, but is found in the cytoplasm in individuals with mild cognitive impairment (MCI) and AD. The function of the 82-kDa ChAT protein is unknown, though recent evidence suggests it has a role in gene expression changes in response to cellular perturbations.

In the present study, we explore whether 82-kDa ChAT is involved in global chromatin organization and an epigenetic response to cytotoxic amyloid-β(Aβ) exposure. We show that 82-kDa ChAT associates with chromatin in human SH-SY5Y neural cells using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), finding that acute exposure of cells to oligomeric Aβ1–42 increases 82-kDa ChAT associations with gene promoters and introns. Following Aβ1–42-exposure, 82-kDa ChAT co-localizes in nuclear aggregates with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 has similar increases in genic associations following Aβ1–42-exposure, and both SATB1 and 82-kDa ChAT associate with synapse-related genes. The 82-kDa ChAT and SATB1 proteins have patterned genomic associations at regions enriched with S/MAR binding motifs, preventing an Aβ1–42-induced increase in an isoform-specific APP mRNA transcript. Finally, we show that 82-kDa ChAT expression during cholinergic differentiation of SH-SY5Y cells increases the steady-state levels of proteins related to synapse formation, resulting in increased neurite complexity.

These results demonstrate that 82-kDa ChAT and SATB1 regulate chromatin organization at S/MARs, resulting in context-dependent gene expression changes in cholinergic cells and increased expression of synapse formation-related proteins during cholinergic differentiation. Cholinergic synapse dysfunction and degeneration is observed early in AD progression and 82-kDa ChAT is mislocalized in AD, therefore the loss of both the epigenetic response to Aβ and gene expression changes related to synapse formation and maintenance may have implications for the etiology or progression of MCI and AD.

Appendix 2- ChAT vehicle gene ontology.xlsx (310 kB)
Appendix 2- ChAT vehicle gene ontology

Appendix 3- ChAT AB gene ontology.xlsx (161 kB)
Appendix 3- ChAT AB gene ontology

Appendix 4- ChAT vehicle and AB gene ontology.xlsx (197 kB)
Appendix 4- ChAT vehicle and AB gene ontology

Appendix 7- SATB1 control gene ontology.xlsx (110 kB)
Appendix 7- SATB1 control gene ontology

Appendix 8- SATB1 AB gene ontology.xlsx (346 kB)
Appendix 8- SATB1 AB gene ontology

Appendix 9- SATB1 control and AB gene ontology.xlsx (119 kB)
Appendix 9- SATB1 control and AB gene ontology

Appendix 10 - ChAT and SATB1 AB gene ontology.xlsx (212 kB)
Appendix 10 - ChAT and SATB1 AB gene ontology

Appendix 11- SATB1 and ChAT vehicle and AB gene ontology.xlsx (43 kB)
Appendix 11- SATB1 and ChAT vehicle and AB gene ontology

Available for download on Monday, October 01, 2018


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