Date of Award

Spring 1994

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Clyde L Denis

Abstract

The yeast CCR4 protein is required for the expression of a number of genes involved in non-fermentative growth, including glucose repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance. It is shown here that CCR4 is able to activate transcription when fused to a heterologous DNA binding domain. The transcriptional activation ability of CCR4, in contrast to many other activators, was glucose regulated. Two activation domains were identified, one of which was glucose responsive and encompassed a glutamine-proline rich region similar to that found in other eukaryotic transcriptional factors. The two transactivation regions, when separated from the leucine-rich repeat and the C-terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C-terminus make contacts that link the activation regions to the proper gene context.

Also reported here is the identification of a protein from mouse (mCAF1) which was capable of interacting with and binding to the yeast CCR4 transcriptional regulatory complex. The mCAF1 protein was shown to share significant similarity with proteins from human, C. elegans, Arabidopsis, and yeast. Both the yeast and C. elegans homologs of mCAF1 were shown to interact with CCR4 in-vivo. Disruption of the yCAF1 gene in yeast gave phenotypes and defects in transcription similar to those seen with disruptions of CCR4. yCAF1 when fused to the LexA DNA binding domain also functioned as a strong activator of transcription in yeast. Immuneprecipitation of yCAF1 revealed that it was complexed with the 185 and 195 kDa species previously shown to associate with CCR4. The binding of CCR4 and yCAF1 to the 185 and 195 Kda proteins was not interdependent. These data indicate that the transcriptional regulatory complex composed of CCR4, the 185 and 195 kDa proteins and yCAF1 make up part of an evolutionarily conserved complex involved in transcriptional control.

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