Investigating the Role of Brachyury (T) in the Control of Chromatin State Within Early Mesodermal Cells
Year: 2017
Type: dissertation
Abstract: The gene Brachyury, T, encodes for the founding member of the T-box family of transcription factors and was previously shown to be crucial for the proper specification and development of the mesodermal germ layer during mouse embryogenesis. In addition to their ability to directly activate and repress transcription by binding to T-box binding sites within target genes, T-box factors were also shown to modulate gene expression by their ability to modify the chromatin environment at target genes through a physical interaction with the MLL/Trithorax family of H3K4 methyltransferases, H3K27 demethylases, and BRG1, a component of the SWI/SNF chromatin remodeling complex of enzymes. This interaction is important for the function of most T-box family members, as multiple mutations in T-box factors which are predicted to disrupt this interaction are found in human patients with congenital developmental disorders. I generated a point mutation within the endogenous T locus of mouse embryonic stem cells, creating an amino acid substitution (Y88A) in the T protein, to determine whether the interaction of T with histone-modifying enzymes is necessary for proper T function during mesodermal development. Mouse embryos expressing the mutant TY88A allele resemble T knockout embryos and lose the ability to maintain T expression, suggesting that the Y88 residue is necessary for proper T function and for maintenance of T expression itself. Gene expression profiling of T-positive mesodermal cells from embryos expressing the TY88A allele revealed that 60% of dysregulated genes were unique to the TY88A mutant when compared to T knockout embryos, suggesting that the amino acid substitution disrupts a critical function of T separate from the maintenance of T expression. Furthermore, gene expression profiling corroborated previous data that T may play a role in hemangioblast commitment, and uncovered a role for T in mammalian erythrocyte specification and development. Genome-wide histone modification profiling in T-positive cells differentiated into mesoderm in vitro revealed that functional interaction with H3K4 methyltransferases and H3K27 demethylases was not disrupted in the TY88A mutant, however, H3K27 acetylation at T binding sites was decreased. Overall, the data in this thesis suggest that the Y88 residue in T is necessary for proper T function during mesodermal specification, and is necessary in the control of H3K27 acetylation at target loci in early mesodermal cells.
Author: Beisaw, Arica
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2583015600