Doctor of Philosophy

dissertationDevelopment of multicellular organisms requires precise coordination of gene expression, regulated by alterations in genomic accessibility and chromatin structure. Changes in chromatin architecture also mediate the transition from plasticity to cell fate commitment. This work describes a candidate RNAi screen in C.elegans designed to identify factors functioning in transcriptional silencing, based on enhancement/suppression of phenotypes associated with mutants in three major transcriptional silencing pathways: mes-3/Polycomb, met-2/SET-DB1, and hpl-1; hpl- 2/HP1. Strikingly, all known members of the Mes/Polycomb pathway of transcriptional repression, including members of PRC2 (mes-2/mes-3/mes-6), mes-4, and set-2 strongly suppress the hpl-1; hpl-2 larval arrest phenotype. A number of additional factors also strongly suppressed hpl-1; hpl-2 arrest, including members of the COMPASS/MLL complex (H3K4me), members of the NuA4 HAT complex, members of SWR1 nucleosome remodeling complex (incorporation of H2A.Z), O-GlcNAc transferase (catalyzes a type of posttranslational modification), a novel factor (T19B4.5), and two proteins involved in DNA repair. Based on the hypothesis that the strong hpl-1; hpl-2 suppression signature indicates association with the Mes pathway, these genes were tested in a number of secondary analyses related to Mes function, including maternal effect sterility, germline iv morphology, H3K27 methylation, interaction in the SynMuv pathway of vulval specification, suppression of mep-1/NuRD larval lethality, and desilencing of tandem arrays in the germline. Phenotypes associated with these assays suggest that hpl-1; hpl-2 suppressors can be divided into two classes based on similarity to either MES-4 or PRC2 phenotypes. Data suggest that the activating complex(es) NuA4/SWR1 interact with MES-4, and may contribute to repression of X linked gene expression. Our analyses also indicate that the COMPASS complex likely functions in the Mes/PcG pathway in two or more forms, one of which is independent of SET-2. Phenotypes associated with SET-2- independent COMPASS complex correlate well with mes-4, while SET-2 may interact more closely with MES/PRC2. This work also demonstrates a novel phenotype associated with the coiled-coil protein T19B4.5. Loss of this factor induces upregulation of let-858::GFP transgenic constructs in the soma, coupled with reduced expression in the germ line. These data suggest that T19B4.5 may modulate both the SynMuv and Mes pathways in a context dependent manner