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Genetic separation of activating and repressing functions of the basic leucine zipper (bZIP) transcription factor Hac1ip on transcription


Nutritional gene regulation is of outstanding interest as a sedentary lifestyle and high calorie diet in western society increases the prevalence of metabolic disorders, e.g. diabetes and obesity. I have shown that a stress signalling pathway from the endo plasmic reticulum (ER) to the nucleus, the unfolded protein response (UPR), transduces a nitrogen signal. The read-out of the UPR, the basic leucine zipper (bZIP) transcription factor Hac1p, repressed transcription of genes activated by nitrogen-starvation.

This repression required the catalytic activity of the RPD3-SIN3 histone deacetylase (HDAC). Hac1p also activates transcription of ER chaperone genes when protein folding in the ER is inhibited. Activation by Hac1p requires the Gcn5p histone acetyltransf erase (HAT). Gcn5p activates transcription by acetylating lysine residues in core histones. Through deacetylation of an overlapping set of lysine residues Rpd3p represses transcription. This strongly suggests that interaction of Hac1p with Gcn5p and Rpd3p is tightly regulated to avoid simultaneous activation of directly opposing transcriptional regulators. To understand the mechanism that controls Hac1p function in response to environmental stimuli I propose to generate Hac1p mutants that are selectively defective in activation or repression only. Site-directed mutagenesis will be used to alter well-known features of this bZIP transcription factor. Random mutagenesis will identify additional mutants. Mutants will be scored in agar plate reporter assays and verified by Northern blotting.

Interaction of these mutants with the HDAC and HAT will be characterised using immuno-precipitation techniques. If separable, this work will identify regulatory mutants of Hac1p, which will enable database searches or genetic suppressor screens for genes that are controlling Hac1p function. This work will elucidate important aspects of how a eukaryotic cell regulates signalling specificity of an inter-organellar signalling pathway.

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Old Elvet
United Kingdom