The mechanism of cyanamide/MMS-induced DDI2/3 expression in Saccharomyces cerevisiae

Abstract

Two genes in Saccharomyces cerevisiae, DDI2 and DDI3, can be highly induced by DNA-damaging agent methyl methanesulfonate (MMS). They share identical coding sequence and their promoter sequences are also highly conserved, which are referred as DDI2/3 in this dissertation. Subsequent studies revealed that DDI2/3 encode a cyanamide (CY) hydratases and are also highly induced by CY. Several approaches identified FZF1, encoding a zinc-finger transcriptional activator, to be responsible for the DDI2/3 induction by MMS and CY. Deletion of FZF1 completely abolished DDI2/3 induction by MMS and CY, whereas controlled overexpression of FZF1 was sufficient to fully induce DDI2/3 expression in the absence of chemical treatment. In addition, our systematic DDI2-lacZ promoter deletion analysis identified both negative and positive cis-acting regulatory elements. The negative elements are in the -709 to -229 region, which is occupied by three nucleosomes. A positive regulatory element named consensus sequence 2 (CS2) is located around -190 to -211, whose internal deletion completely abolished CY- or MMS-induced DDI2 reporter gene expression. Using the 27-bp CS2 sequence as a probe, electrophoretic mobility shift assays revealed sequence-specific DNA-protein interactions from CY-induced or FZF1-overexpressed cells, and this interaction was absent in the fzf1 mutant. In addition, mass spectrometry detected methylations of Fzf1 on K70 and K107 residues after MMS treatment, as well as CY modifications on K122 and K184 residues after CY treatment. Among amino-acid substitutions of the above Lys residues, Fzf1-K70A completely abolished the MMS induction and decreased CY induction. Moreover, CY and MMS treatments significantly reduced cellular histone levels, leading to a working model that CY and MMS play dual roles in inducing DDI2/3 in yeast cells. This research project advances our knowledge of gene regulation in response to two simple chemicals. It reveals two rather unusual molecular mechanisms that jointly induce the massive expression of DDI2/3. Understanding the mechanism of CY- and MMS-induced DDI2/3 expression will lead to a comprehensive understanding of eukaryotic response to environmental stresses.