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Theses Doctoral

Cell fate restriction in Caenorhabditis elegans is orchestrated by precise chromatin organization and transcription factor activity

Patel, Tulsi

The plasticity of cells in a multicellular organism is progressively lost during differentiation. This loss is reflected in studies involving the ectopic misexpression of fate-specifying or terminal selector transcription factors (TFs). These TFs can efficiently activate target genes in undifferentiated cells, but lose this ability as cells differentiate. While this phenomenon of cell fate restriction is widely observed, the mechanisms orchestrating it are poorly understood. In this thesis, I have used the ubiquitous overexpression of Zn-finger-TF CHE-1 as a tool to understand the mechanisms that restrict cell fate in Caenorhabditis elegans. When CHE-1 is ubiquitously expressed at embryonic stages, it activates target gene expression in many cell types, while in adults it can only act in a few neurons. To uncover factors that inhibit plasticity of all other adult cells, I first performed an RNAi screen against chromatin-associated factors. Using this approach I found that the removal of either the PRC2 complex, which deposits the H3K27me3 mark, or loss of proteins that indirectly regulate domains of H3K27me3, allows CHE-1 and two other terminal selector TFs to activate target genes in the germline. These data show that the correct distribution of H3K27me3 is crucial for the restriction of germ cell fate. I next took a candidate approach to identify genes that regulate fate restriction in other cell types. We hypothesized that terminal selector TFs themselves, in addition to specifying cellular identity by controlling large gene sets, may also act to inhibit plasticity. To test this, I first assayed the activity of CHE-1 in mutants of COE-TF unc-3, the terminal selector for a subset of cholinergic motor neurons (MNs). I found that in contrast to wildtype MNs, unc-3 mutant MNs remain plastic as CHE-1 can induce expression of target genes in these cells even at the adult stage. This phenotype is also observed in four of six additional terminal selector mutants tested. I further found that the removal of met-2, a protein required for H3K9 methylation, or mes-2, a PRC2 component, also makes differentiated cholinergic MNs amenable to the activity of CHE-1. Preliminary evidence suggests that met-2 may act in the same pathway as unc-3. These results raise the exciting possibility that selector TFs play a role in restricting cell fate by organizing the heterochromatin domains in differentiated cells. Overall, in this work I provide functional evidence to show that specific chromatin-modifying enzymes restrict the fate of germ cells and that both fate-specifying TFs and chromatin-modifying enzymes are required for the fate restriction in neurons.

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More About This Work

Academic Units
Genetics and Development
Thesis Advisors
Hobert, Oliver
Degree
Ph.D., Columbia University
Published Here
May 3, 2016
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