Abstract: Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP.
Abstract: NC2 is a heterodimeric regulator of transcription that plays both positive and negative roles in vivo. Here we show that the alpha and beta subunits of yeast NC2 are not always associated in a tight complex. Rather, their association is regulated, in particular by glucose depletion. Indeed, stable NC2 alpha/beta complexes can only be purified from cells after the diauxic shift when glucose has been depleted from the growth medium. In vivo, the presence of NC2 alpha, but not NC2 beta, at promoters generally correlates with the presence of TBP and transcriptional activity. In contrast, increased presence of NC2 beta relative to TBP correlates with transcriptional repression. NC2 is regulated by phosphorylation. We found that mutation of genes encoding casein kinase II (CKII) subunits as well as potential CKII phosphorylation sites in NC2 alpha and beta affected gene repression. Interestingly, NC2-dependent repression in the phosphorylation site mutants was only perturbed in high glucose when NC2 beta and NC2 alpha are not associated, but not after the diauxic shift when NC2 alpha and beta form stable complexes. Thus, the separation of NC2 alpha and beta function indicated by these mutants also supports the existence of multiple NC2 complexes with different functions in transcription.
Abstract: When grown in the presence of sunflower cell walls, Sclerotinia sclerotiorum, an ubiquitous necrotrophic fungus, secretes several acid proteases including a non-aspartyl protease. The gene acp1, encoding an acid protease, has been cloned and sequenced. The intronless ORF encodes a preproprotein of 252 aa and a mature protein of 200 residues. In vitro expression of acp1 is subject to several transcriptional regulatory mechanisms. Expression induced by plant cell-wall proteins is controlled by both carbon and nitrogen catabolite repression. Glucose on its own represses acp1 expression while ammonium repression requires the simultaneous presence of a carbon source. Ambient pH higher than pH 5 overrides induction resulting in full repression of acp1. These transcriptional regulatory mechanisms and the presence of several motifs in the promoter of acp1 that may encode binding sites for the regulators CREA, AREA and PacC suggest the involvement of these regulators in the control of acp1 expression. acp1 is expressed in planta during sunflower cotyledon infection. Expression is low at the beginning of infection but increases suddenly at the stage of necrosis spreading. Comparison of in vitro and in planta acp1 expression suggests that glucose and nitrogen starvation together with acidification can be considered as key factors controlling Scl. sclerotiorum gene expression during pathogenesis.
